Nitrile hydratase

ABSTRACT

Provided is an improved nitrile hydratase with improved catalytic activity. Also provided are DNA for coding the improved nitrile hydratase, a recombinant vector that contains the DNA, a transformant that contains the recombinant vector, nitrile hydratase acquired from a culture of the transformant, and a method for producing the nitrile hydratase. Also provided is a method for producing an amide compound that uses the culture or a processed product of the culture. The improved nitrile hydratase contains an amino acid sequence represented by SEQ ID NO: 50 (GX 1 X 2 X 3 X 4 DX 5 X 6 R) in a beta subunit, and is characterized in that X 4  is an amino acid selected from a group comprising cysteine, aspartic acid, glutamic acid, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, serine and threonine.

This application is a divisional of U.S. application Ser. No. 14/853,086filed Sep. 14, 2015, allowed, which is a divisional of U.S. applicationSer. No. 14/124,555 filed Dec. 6, 2013, U.S. Pat. No. 9,193,966, whichis a National Stage of PCT/JP12/003745 filed Jun. 7, 2012 and claims thebenefit of JP 2011-127466 filed Jun. 7, 2011, JP 2011-144378 filed Jun.29, 2011 and JP 2011-145061 filed Jun. 30, 2011.

TECHNICAL FIELD

The present invention relates to improving a nitrile hydratase(mutation) and its production method. Moreover, the present inventionrelates to genomic DNA that encodes the enzyme, a recombinant vectorcontaining the genomic DNA, a transformant containing the recombinantvector, and a method for producing an amide compound.

DESCRIPTION OF BACKGROUND ART

In recent years, a nitrile hydratase was found, which is an enzymehaving nitrile hydrolysis activity that catalyses the hydration of anitrile group to its corresponding amide group. Also, methods aredisclosed to produce corresponding amide compounds from nitrilecompounds using the enzyme or a microbial cell or the like containingthe enzyme. Compared with conventional chemical synthetic methods, suchmethods are known by a high conversion or selectivity rate from anitrile compound to a corresponding amide compound.

Examples of microorganisms that produce a nitrile hydratase are thegenus Corynebacterium, genus Pseudomonas, genus Rhodococcus, genusRhizobium, genus Klebsiella, genus Pseudonocardia and the like. Amongthose, Rhodococcus rhodochrous strain J1 has been used for industrialproduction of acrylamides, and its usefulness has been verified.Furthermore, a gene encoding a nitrile hydratase produced by strain J1has been identified (see patent publication 1).

Meanwhile, introducing a mutation into a nitrile hydratase has beenattempted not only to use a nitrile hydratase isolated from a naturallyexisting microorganism or its gene, but also to change its activity,substrate specificity, Vmax, Km, heat stability, stability in asubstrate, stability in a subsequent product and the like of a nitrilehydratase. Regarding the nitrile hydratase in Pseudonocardia thermophilaJCM 3095, from its conformational data, sites relating to the substratespecificity or thermal stability are anticipated, and mutant enzymeswith modified substrate specificity were obtained (see patentpublications 2˜4). Also, nitrile hydratase genes with improved heatresistance and amide-compound resistance have been produced by theinventors of the present invention (see patent publications 5˜9).

To produce acrylamide for industrial applications using enzymeproduction methods, it is useful to develop a nitrile hydratase withimproved catalytic activity when production costs such as catalyst costsare considered. Developing enzymes with improved activity is especiallydesired so as to achieve a reduction in the enzyme amount for reactionsand in production costs or the like.

PRIOR ART PUBLICATION Patent Publication

-   Patent publication 1: Japanese patent publication 3162091-   Patent publication 2: International publication pamphlet    WO2004/056990-   Patent publication 3: Japanese laid-open patent publication    2004-194588-   Patent publication 4: Japanese laid-open patent publication    2005-16403-   Patent publication 5: International publication pamphlet    WO2005/116206-   Patent publication 6: Japanese laid-open patent publication    2007-143409-   Patent publication 7: Japanese laid-open patent publication    2007-43910-   Patent publication 8: Japanese laid-open patent publication    2008-253182 Patent publication 9: Japanese laid-open patent    publication 2010-172295

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The objective of the present invention is to improve a nitrile hydrataseso as to provide an improved nitrile hydratase with enhanced catalyticactivity. Another objective of the present invention is to provide anitrile hydratatse collected from DNA encoding such an improved nitrilehydratase, a recombinant vector containing the DNA, a transformantcontaining the recombinant vector, and a culture of the transformant, aswell as a method for producing such a nitrile hydratase. Yet anotherobjective of the present invention is to provide a method for producingan amide compound using the culture or the processed product of theculture.

Solutions to the Problems

The inventors of the present invention have conducted extensive studiesto solve the above problems. As a result, in the amino acid sequence ofa nitrile hydatase, the inventors have found that a protein in which aspecific amino-acid residue is substituted with another amino-acidresidue has nitrile hydratase activity and exhibits enhanced catalyticactivity. Accordingly, the present invention is completed.

Namely, the present invention is described as follows.

(1) An improved nitrile hydratase characterized by at least one of thefollowing (a)˜(e):

(a) in the β subunit, a nitrile hydratase contains an amino-acidsequence as shown in SEQ ID NO: 50 below

(SEQ ID NO: 50) GX₁X₂X₃X₄DX₅X₆R(G is glycine, D is aspartic acid, R is arginine, and X₁, X₂, X₃, X₅ andX₆ each independently indicate any amino-acid residue), in which X₄ isan amino acid selected from among cysteine, aspartic acid, glutamicacid, histidine, isoleucine, lysine, methionine, asparagine, proline,glutamine, serine and threonine;

(b) in the β subunit, a nitrile hydratase contains an amino-acidsequence as shown in SEQ ID NO: 81 below

(SEQ ID NO: 81) WEX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈D (W is tryptophan, E is glutamic acid, D is aspartic acid, and X₁˜X₆, andX₈˜X₁₈ each independently indicate any amino-acid residue), in which X₇is an amino acid selected from among alanine, valine, aspartic acid,threonine, phenylalanine, isoleucine and methionine;

(c) in the α subunit, a nitrile hydratase contains an amino-acidsequence as shown in SEQ ID NO: 119 below

(SEQ ID NO: 119) AX₁X₂X₃X₄GX₅X₆GX₇X₈(A is alanine, G is glycine, and X₁˜X₇ each independently indicate anyamino-acid residue), in which X₈ is an amino acid selected from amongalanine, leucine, methionine, asparagine, cysteine, aspartic acid,glutamic acid, phenylalanine, glycine, histidine, lysine, proline,arginine, serine, threonine and tryptophan;

(d) in the α subunit, a nitrile hydratase has the amino-acid sequence asshown in SEQ ID NO: 132 below,

(SEQ ID NO: 132) AX₁X₂X₃X₄GX₅X₆GX₇Q(A is alanine, G is glycine, Q is glutamine, and X₁˜X₆ eachindependently indicate any amino-acid residue), in which X₇ issubstituted with an amino acid different from that in a wild type;

(e) in the α subunit, a nitrile hydratase has the amino-acid sequence asshown in SEQ ID NO: 136 below

(SEQ ID NO: 136) AX₁X₂X₃X₄GX₅X₆GX₇QX₈X₉(A is alanine, G is glycine, Q is glutamine, and X₁˜X₈ eachindependently indicate any amino-acid residue), in which X₉ issubstituted with an amino acid different from that in a wild type.

(2) The improved nitrile hydratase described in (1), characterized inthat X₂ in SEQ ID NO: 50 is S (serine).

(3) The improved nitrile hydratase described in (1), characterized inthat X₁ is I (isoleucine), X₂ is S (serine), X₃ is W (tryptophan), X₅ isK (lysine), and X₆ is S (serine) in SEQ ID NO: 50.

(4) The improved nitrile hydratase described in any of (1)˜(3), havingan amino-acid sequence as shown in SEQ ID NO: 51 that includes theamino-acid sequence as shown in SEQ ID NO: 50.

(5) The improved nitrile hydratase described in (1), characterized inthat X₁₄ in SEQ ID NO: 81 is G (glycine).

(6) The improved nitrile hydratase described in (1), characterized inthat X₁ is G (glycine), X₂ is R (arginine), X₃ is T (threonine), X₄ is L(leucine), X₅ is S (serine), X₆ is I (isoleucine), X₈ is T (threonine),X₉ is W (tryptophan), X₁₀ is M (methionine), X₁₁ is H (histidine), X₁₂is L (leucine), X₁₃ is K (lysine), and X₁₄ is G (glycine) in SEQ ID NO:81.

(7) The improved nitrile hydratase described in any of (1), (5) and (6),having an amino-acid sequence as shown in SEQ ID NO: 82 that includesthe amino-acid sequence as shown in SEQ ID NO: 81.

(8) The improved nitrile hydratase described in (1), characterized inthat X₁ is M (methionine), X₂ is A (alanine), X₃ is S (serine), X₄ is L(leucine), X₅ is Y (tyrosine), X₆ is A (alanine) and X₇ is E (glutamicacid) in SEQ ID NO: 119.

(9) The improved nitrile hydratase described in (1) or (8), having anamino-acid sequence as shown in SEQ ID NO: 120 that includes theamino-acid sequence as shown in SEQ ID NO: 119.

(10) The improved nitrile hydratase described in (1), characterized bycontaining the amino-acid sequence of the α subunit as shown in SEQ IDNO: 132, in which X₇ is an amino acid selected from among cysteine,phenylalanine, histidine, isoleucine, lysine, methionine, glutamine,arginine, threonine and tyrosine.

(11) The improved nitrile hydratase described in (1) or (10),characterized in that X₁ is M (methionine), X₂ is A (alanine), X₃ is S(serine), X₄ is L (leucine), X₅ is Y (tyrosine), and X₆ is A (alanine)in SEQ ID NO: 132.

(12) The improved nitrile hydratase described in (1), (10) or (11),having an amino-acid sequence as shown in SEQ ID NO: 131 that includesthe amino-acid sequence as shown in SEQ ID NO: 132.

(13) The improved nitrile hydratase described in (1), characterized bycontaining an amino-acid sequence of the α subunit as shown in SEQ IDNO: 136, in which X₉ is an amino acid selected from among cysteine,glutamic acid, phenylalanine, isoleucine, asparagine, glutamine, serineand tyrosine.

(14) The improved nitrile hydratase described in (1) or (13),characterized in that X₁ is M (methionine), X₂ is A (alanine), X₃ is S(serine), X₄ is L (leucine), X₅ is Y (tyrosine), X₆ is A (alanine), X₇is E (glutamic acid), and X₈ is A (alanine) in SEQ ID NO: 136.

(15) The improved nitrile hydratase described in (1), (13) or (14),having an amino-acid sequence as shown in SEQ ID NO: 135 that includesthe amino-acid sequence as shown in SEQ ID NO: 136.

(16) The improved nitrile hydratase described in any one of (1) to (15)is a nitrile hydratase derived from Rhodococcus bacterium or Nocardiabacterium.

(17) DNA encoding the improved nitrile hydratase described in any one of(1) to (16).

(18) DNA hybridized with the DNA described in (17) under stringentconditions.

(19) A recombinant vector containing the DNA described in (17) or (18).

(20) A transformant containing the recombinant vector described in (19).

(21) A nitrile hydratase collected from a culture obtained by incubatingthe transformant described in (20).

(22) A method for producing a nitrile hydratase, such a methodcharacterized by incubating the transformant described in (20) and bycollecting the nitrile hydratase from the obtained culture.

(23) A method for producing an amide compound, such a methodcharacterized by bringing a nitrile compound into contact with aculture, or a processed product of the culture, obtained by incubatingthe improved nitrile hydratase described in any of (1)˜(16) or thetransformant described in (20).

Effects of the Invention

According to the present invention, a novel improved (mutant) nitrilehydratase is obtained to have enhanced catalytic activity. The improvednitrile hydratase with enhanced catalytic activity is very useful toproduce amide compounds at a high yield.

According to the present invention, an improved nitrile hydratase andits production method are provided; such a nitrile hydratase is obtainedfrom genomic DNA encoding the improved nitrile hydratase, a recombinantvector containing the genomic DNA, a transformant containing therecombinant vector and a culture of the transformant. Also provided bythe present invention is a method for producing an amide compound usingthe protein (improved nitrile hydratase) and the culture or a processedproduct of the culture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing the structure of plasmid pSJ034;

FIG. 2-1 is a list showing the alignment results in β subunits of knownnitrile hydratases;

FIG. 2-2 is a list showing the alignment results in β subunits of knownnitrile hydratases;

FIG. 3 shows the amino-acid sequence of the β subunit identified as SEQID NO: 51 related to the present invention;

FIG. 4 is a photograph showing results of SDS-PAGE;

FIG. 5 is a view showing the structure of plasmid pER855A;

FIG. 6-1 is a list showing amino-acid sequences (part of N-terminalside) of β subunits in wild-type nitrile hydratases derived from variousmicroorganisms;

FIG. 6-2 is a list showing amino-acid sequences (part of C-terminalside) subsequent to the amino-acid sequences in FIG. 6-1;

FIG. 7 shows the amino-acid sequence in the β subunit identified as SEQID NO: 82 related to the present invention;

FIG. 8-1 is a list showing amino-acid sequences (part of N-terminalside) in α subunits of nitrile hydratases derived from variousmicroorganisms;

FIG. 8-2 is a list showing amino-acid sequences subsequent to theamino-acid sequences in FIG. 8-1;

FIG. 9 shows the amino-acid sequence in the α subunit identified as SEQID NO: 121 related to the present invention;

FIG. 10-1 is a list showing amino-acid sequences (part of N-terminalside) in α subunits of nitrile hydratases derived from variousmicroorganisms;

FIG. 10-2 is a list showing amino-acid sequences the same as in FIG.2-1, and shows the sequences subsequent to the amino-acid sequences inFIG. 10-1;

FIG. 11 shows the amino-acid sequence in the α subunit identified as SEQID NO: 131 related the present invention;

FIG. 12-1 is a list showing amino-acid sequences (part of N-terminalside) in α subunits of nitrile hydratases derived from variousmicroorganisms;

FIG. 12-2 is a list showing amino-acid sequences subsequent to theamino-acid sequences in FIG. 12-1; and

FIG. 13 shows the amino-acid sequence in the α subunit identified as SEQID NO: 135 related to the present invention.

MODE TO CARRY OUT THE INVENTION

In the following, the present invention is described in detail.

1. Nitrile Hydratase

(a) Known Nitrile Hydratase

The improved nitrile hydratase of the present invention is obtained bymodifying a known nitrile hydratase and is not limited to being derivedfrom any specific type. For example, those registered as nitrilehydratases in the GenBank database provided by the U.S. National Centerfor Biotechnology Information (NCBI), or those described as nitrilehydratases in publications, may be referred to for a use. Examples ofsuch nitrile hydratases are those described in patent publications 5˜9(which are incorporated by reference in the present application).Nitrile hydratases in patent publications 5˜9 have heat resistance andacrylamide resistance, and by employing amino-acid substitutionsaccording to the present invention, enhanced catalytic activity isfurther added to their properties. In particular, nitrile hydrataseshaving amino-acid sequences shown in SEQ ID NOs: 53˜57 are listed asreference.

Furthermore, by introducing a mutation from the gene encoding theamino-acid sequences described above using a well-known method, and byevaluating and screening mutant enzymes which have desired properties,improved enzymes with further enhanced activity are achieved. Inparticular, nitrile hydratases with amino-acid sequences shown in SEQ IDNOs: 58˜61 are listed.

A “nitrile hydratase” has a conformation formed with α and β subunitdomains, and contains a non-heme iron atom or a non-corrin cobalt atomas a prosthetic molecule. Such a nitrile hydratase is identified andreferred to as an iron-containing nitrile hydratase or acobalt-containing nitrile hydratase.

An example of an iron-containing nitrile hydratase is such derived fromRhodococcus N-771 strain. The tertiary structure of such aniron-containing nitrile hydratase has been identified by X-ray crystalstructural analysis. The enzyme is bonded with non-heme iron via fouramino-acid residues in a cysteine cluster (Cys-Ser-Leu-Cys-Ser-Cys) (SEQID NO: 48) forming the active site of the α subunit.

As for a cobalt-containing nitrile hydratase, examples are those derivedfrom Rhodococcus rhodochrous J1 strain (hereinafter may be referred toas “J1 strain”) or derived from Pseudonocardia thermophila.

A cobalt-containing nitrile hydratase derived from the J1 strain isbound with a cobalt atom via a site identified as a cysteine cluster(Cys-Thr-Leu-Cys-Ser-Cys) (SEQ ID NO: 49) that forms the active site ofthe α subunit. In the cysteine cluster of a cobalt-containing nitrilehydratase derived from Pseudonocardia thermophila, cysteine (Cys) atposition 4 from the upstream side (N-terminal side) of the cysteinecluster derived from the J1 strain is cysteine sulfinic acid (Csi), andcysteine (Cys) at position 6 from the furthermost downstream side(C-terminal side) of the cysteine cluster derived from the J1 strain iscysteine sulfenic acid (Cse).

As described above, a prosthetic molecule is bonded with a siteidentified as cysteine clusters “C(S/T)LCSC” (SEQ ID NO: 48, 49) in theα subunit. Examples of a nitrile hydratase containing a binding sitewith such a prosthetic molecule are those that have amino-acid sequencesand are encoded by gene sequences derived from the following:Rhodococcus rhodochrous J1 (FERM BP-1478), Rhodococcus rhodochrous M8(SU 1731814), Rhodococcus rhodochrous M33 (VKM Ac-1515D), Rhodococcusrhodochrous ATCC 39484 (JP 2001-292772), Bacillus smithii (JPH9-248188), Pseudonocardia thermophila (JP H9-275978), or Geobacillusthermoglucosidasius.

On the other hand, the β-subunit is thought to be attributed tostructural stability.

For example, in the α subunit derived from Rhodococcus rhodochrous J1strain (FERM BP-1478), its amino-acid sequence is shown as SEQ ID NO: 4,and its base sequence is shown as SEQ ID NO: 3. Also, in the 1 subunit,its amino-acid sequence is shown as SEQ ID NO: 2, its base sequence isshown as SEQ ID NO: 1 and its accession number is “P21220.” In addition,in Rhodococcus rhodochrous M8 (SU 1731814), the accession number of theα subunit is “ATT 79340” and the accession number of the β subunit is“AAT 79339.”

The accession number of the nitrile hydratase gene derived fromRhodococcus pyridinivorans MW3 is “AJ582605,” and the accession numberof the nitrile hydratase gene derived from Rhodococcus pyridinivoransS85-2 is “AJ582605.” The nitrile hydratase gene of Rhodococcus ruber RH(CGMCC No. 2380) is described in CN 101463358. Moreover, the accessionnumber of the nitrile hydratase gene derived from Nocardia YS-2002 is“X86737,” and the accession number of the nitrile hydratase gene derivedfrom Nocardia sp. JBRs is “AY141130.”

(b-1) Improved Nitrile Hydratase (β48)

FIGS. 2-1 and 2-2 show the alignments of amino-acid sequences (inone-letter code) in β-subunits of known nitrile hydratases derived fromvarious microorganisms. FIGS. 2-1 and 2-2 each show amino-acid sequencesin sequence ID numbers 2, 5˜12, and 42˜47 of amino-acid sequences fromthe top.

Furthermore, the improved nitrile hydratase of the present inventionincludes examples in which one or more (for example, 1˜10, preferred tobe approximately 1˜5) amino-acid residues are deleted, substitutedand/or added in the amino-acid sequences of known nitrile hydratases,excluding the amino-acid sequence identified as SEQ ID NO: 50.

An example of the improved nitrile hydratase of the present inventionhas an amino-acid sequence identified as SEQ ID NO: 51 in the β subunitas shown in FIG. 3. Here, the amino-acid sequence shown as SEQ ID NO: 50is located at positions 44˜52 counted from the N-terminal.

According to an embodiment of the example above, in the improved nitrilehydratase that has the amino-acid sequence as shown in SEQ ID NO: 51,X₁, X₂, X₃, X₅, and X₆ each independently indicate any amino-acidresidue, and X₄ is an amino acid selected from among cysteine, asparticacid, glutamic acid, histidine, isoleucine, lysine, methionine,asparagine, proline, glutamine, serine and threonine.

In addition, according to another embodiment, in the improved nitrilehydratase that has the amino-acid sequence as shown in SEQ ID NO: 51,X₁, X₃, X₅, and X₆ each independently indicate any amino-acid residue,X₂ is S (serine), and X₄ is an amino acid selected from among cysteine,aspartic acid, glutamic acid, histidine, isoleucine, lysine, methionine,asparagine, proline, glutamine, serine and threonine.

Moreover, according to yet another embodiment, in the improved nitrilehydratase that has the amino-acid sequence as shown in SEQ ID NO: 51, X₁is I (isoleucine), X₂ is S (serine), X₃ is W (tryptophan), and X₅ is K(lysine), X₆ is S (serine), and X₄ is an amino acid selected from amongcysteine, aspartic acid, glutamic acid, histidine, isoleucine, lysine,methionine, asparagine, proline, glutamine, serine and threonine.

Another example of the improved nitrile hydratase of the presentinvention is as follows: in the amino-acid sequence of a known nitrilehydratase identified as SEQ ID NO: 2, the amino-acid residue(tryptophan) at position 48 of the β subunit is substituted withcysteine, aspartic acid, glutamic acid, histidine, isoleucine, lysine,methionine, asparagine, proline, glutamine, serine or threonine.

Modes of such amino-acid substitutions are denoted, for example, asWβ48C, Wβ48D, Wβ48E, Wβ48H, Wβ48I, ω348K, Wβ48M, Wβ48N, Wβ48P, Wβ48Q,Wβ48S or Wβ48T. Amino acids are identified by a single-letter alphabeticcode. The letter to the left of the numeral showing the number ofamino-acid residues counted from the terminal to the substitutedposition (for example, “48”) represents the amino acid in a one-lettercode before substitution, and the letter to the right represents theamino acid in a one-letter code after substitution.

In particular, when the amino-acid sequence of the β subunit as shown inSEQ ID NO: 2 is denoted as “Wβ48C” in the improved nitrile hydratase,the abbreviation means that, in the amino-acid sequence of the β subunit(SEQ ID NO: 2), tryptophan (W) at position 48 counted from theN-terminal amino-acid residue (including the N-terminal amino-acidresidue itself) is substituted with cysteine (C).

Modes of amino acid substitutions in more preferred embodiments of theimproved nitrile hydratase according to the present invention are shownas the following 1˜12:

1. Wβ48C,

2. Wβ48D,

3. Wβ48E,

4. Wβ48H,

5. Wβ48I,

6. Wβ48K,

7. Wβ48M,

8. Wβ48N,

9. Wβ48P,

10. Wβ48Q,

11. Wβ48S, and

12. Wβ48T.

Preferred embodiments of base substitutions to cause the aboveamino-acid substitutions are shown below.

Wβ48C: a base sequence TOG (at positions 142˜144 in SEQ ID NO: 1) ispreferred to be substituted with TGC (TGG-TGC).

Wβ48D: a base sequence TGG (at positions 142˜144 in SEQ ID NO: 1) ispreferred to be substituted with GAC (TGG→GAC).

Wβ48E: a base sequence TGG (at positions 142˜144 in SEQ ID NO: 1) ispreferred to be substituted with GAG (TGG→GAG).

Wβ48F: a base sequence TGG (at positions 142˜144 in SEQ ID NO: 1) ispreferred to be substituted with TTC (TGG→TTC).

W 348H: a base sequence TGG (at positions 142˜144 in SEQ ID NO: 1) ispreferred to be substituted with CAC (TGG→CAC).

Wβ48I: a base sequence TGG (at positions 142˜144 in SEQ ID NO: 1) ispreferred to be substituted with ATC (TGG→ATC).

Wβ48K: a base sequence TOG (at positions 142˜144 in SEQ ID NO: 1) ispreferred to be substituted with AAG (TGG→AAG).

Wβ48M: a base sequence TGG (at positions 142˜144 in SEQ ID NO: 1) ispreferred to be substituted with ATG (TGG→ATG).

Wβ48N: a base sequence TGG (at positions 142˜144 in SEQ ID NO: 1) ispreferred to be substituted with AAC (TGG→AAC).

Wβ48P: a base sequence TGG (at positions 142˜144 in SEQ ID NO: 1) ispreferred to be substituted with CCG (TGG→CCG).

Wβ48Q: a base sequence TGG (at positions 142˜144 in SEQ ID NO: 1) ispreferred to be substituted with CAG (TGG→CAG).

Wβ48S: a base sequence TGG (at positions 142˜144 in SEQ ID NO: 1) ispreferred to be substituted with TCC (TGG→TCC).

Wβ48T: a base sequence TGG (at positions 142˜144 in SEQ ID NO: 1) ispreferred to be substituted with ACC (TGG→ACC).

(b-2) Improved Nitrile Hydratase (β37)

FIGS. 6-1 and 6-2 show the alignments of amino-acid sequences (in theone-letter code) in β-subunits of known nitrile hydratases derived fromvarious microorganisms. FIGS. 6-1 and 6-2 each show amino-acid sequencesin sequence ID numbers 2, 5˜12, and 42˜49 of amino-acid sequences fromthe top.

Furthermore, the improved nitrile hydratase of the present inventionincludes examples in which one or more (for example, 1˜10, preferred tobe approximately 1˜5) amino-acid residues are deleted, substitutedand/or added in the amino-acid sequences of known nitrile hydratases,excluding the amino-acid sequence identified as SEQ ID NO: 81.

An example of the improved nitrile hydratase of the present inventionhas an amino-acid sequence identified as SEQ ID NO: 82 in the β subunitas shown in FIG. 7. Here, the amino-acid sequence shown in SEQ ID NO: 81is located at positions 29˜49 counted from the N-terminal.

According to an embodiment, in the improved nitrile hydratase that hasthe amino-acid sequence shown in SEQ ID NO: 82, X₁˜X₆ and X₈˜X₁₈ eachindependently indicate any amino-acid residue, and X₇ is an amino acidselected from among alanine, aspartic acid, threonine, phenylalanine,isoleucine and methionine.

According to another embodiment, in the improved nitrile hydratase thathas the amino-acid sequence shown in SEQ ID NO: 82, X₁˜X₆, X₈˜X₁₃ andX₁₅˜X₁₈, each independently indicate any amino-acid residue, X₄ is G(glycine), and X₇ is an amino acid selected from among alanine, valine,aspartic acid, threonine, phenylalanine, isoleucine and methionine.

According to yet another embodiment, in the improved nitrile hydratasethat has the amino-acid sequence as shown in SEQ ID NO: 82, X₁₅˜X₁₈ eachindependently indicate any amino-acid residue, X₁ is G (glycine), X₂ isR (arginine), X₃ is T (threonine), X₄ is L (leucine), X₅ is S (serine),X₆ is I (isoleucine), X₈ is T (threonine), X₉ is W (tryptophan), X₁₀ isM (methionine), X₁₁ is H (histidine), X₁₂ is L (leucine), X₁₃ is K(lysine), X₁₄ is G (glycine), X₇ is an amino acid selected from amongalanine, valine, aspartic acid, threonine, phenylalanine, isoleucine andmethionine.

Another example of the improved nitrile hydratase of the presentinvention is as follows: in the amino-acid sequence of a known nitrilehydratase identified as SEQ ID NO: 2, the amino-acid residue (leucine)at position 37 of the 1 subunit is substituted with alanine, valine,aspartic acid, threonine, phenylalanine, isoleucine or methionine.

Modes of such amino-acid substitutions are denoted, for example, asLβ37A, Lβ37D, Lβ37F, Lβ37I, Lβ37M, Lβ37T or Lβ37V. Amino acids areidentified by a single-letter alphabetic code. The letter to the left ofthe numeral showing the number of amino-acid residues counted from theterminal to the substituted position (for example, “37”) is the aminoacid in the one-letter code before substitution, and the letter to theright represents the amino acid in the one-letter code aftersubstitution.

In particular, when the amino-acid sequence of the β subunit (SEQ ID NO:2) identified as SEQ ID NO: 2 is denoted as “Lβ37A” in the improvednitrile hydratase, the abbreviation means that, in the amino-acidsequence of the β subunit (SEQ ID NO: 2), leucine (L) at position 37counted from the N-terminal amino-acid residue (including the N-terminalamino-acid residue itself) is substituted with alanine (A).

Modes of amino acid substitutions in more preferred embodiments of theimproved nitrile hydratase according to the present invention are shownas the following 1˜7:

1. Lβ37A,

2. Lβ37D,

3. Lβ37F,

4. Lβ37I,

5. Lβ37M,

6. Lβ37T and

7. Lβ37V.

Preferred embodiments of base substitutions to cause the aboveamino-acid substitutions are shown in Table 1 below.

TABLE 1 amino-acid substitution base substitution Lβ37A Base sequenceCTG (positions at 109~111 in SEQ ID NO: 1) is preferred to besubstituted with GCA, GCC, GCG or GCT. Especially preferred to besubstituted is C at position 109 with G, T at position 110 with C, and Gat position 111 with C (CTG→GCC). Lβ37D Base sequence CTG (positions at109~111 in SEQ ID NO: 1) is preferred to be substituted with GAC or GAT.Especially preferred to be substituted is C at position 109 with G, T atposition 110 with A, and G at position 111 with C (CTG→GAC). Lβ37F Basesequence CTG (positions at 109~111 in SEQ ID NO: 1) is preferred to besubstituted with TTC or TTT. Especially preferred to be substituted is Cat position 109 with T and G at position 111 with C (CTG→TTC). Lβ37IBase sequence CTG (positions at 109~111 in SEQ ID NO: 1) is preferred tobe substituted with ATT, ATC or ATA. Especially preferred to besubstituted is C at position 109 with A and G at position 111 with C(CTG→ATC). Lβ37M Base sequence CTG (positions at 109~111 in SEQ IDNO: 1) is preferred to be substituted with ATG. Especially preferred tobe substituted is C at position 109 with A (CTG→ATG). Lβ37T Basesequence CTG (positions at 109~111 in SEQ ID NO: 1) is preferred to besubstituted with ACA, ACC, ACG or ACT. Especially preferred to besubstituted is C at position 109 with A, T at position 110 with C and Gat position 111 with C (CTG→ACC). Lβ37V Base sequence CTG (positions at109~111 in SEQ ID NO: 1) is preferred to be substituted with GTA, GTC,GTG or GTT. Especially preferred to be substituted is C at position 109with G and G at position 111 with C (CTG→GTC).(b-3) Improved Nitrile Hydratase (α83)

FIGS. 8-1 and 8-2 show amino-acid sequence alignments (in one-lettercode) in α-subunits of known nitrile hydratases derived from variousmicroorganisms. FIGS. 8-1 and 8-2 each show amino-acid sequences insequence ID numbers 4, 105˜108, 121, 109, 110, 112, 111, 122˜124, 113,114, 125 from the top.

Furthermore, the improved nitrile hydratase of the present inventionincludes examples in which one or more (for example, 1˜10, preferred tobe approximately 1˜5) amino-acid residues are deleted, substitutedand/or added in amino-acid sequences of known nitrile hydratases,excluding the amino-acid sequence identified as SEQ ID NO: 119. Examplesof such a nitrile hydratase are described in patent publications 5˜9(the contents are incorporated by reference into the presentapplication). Nitrile hydratases in patent publication 5˜9 each exhibitheat resistance and acrylamide resistance. Moreover, as a result ofamino-acid substitutions of the present invention, enhanced catalyticactivity is further added to their properties.

An example of the improved nitrile hydratase of the present inventionhas an amino-acid sequence as shown in SEQ ID NO: 120 in the α subunitas shown in FIG. 9. Here, an amino-acid sequence shown in SEQ ID NO: 119is located at positions 73˜83 counted from the N-terminal.

According to an embodiment, in the improved nitrile hydratase that hasthe amino-acid sequence shown in SEQ ID NO: 120, X₁˜X₇ eachindependently indicate any amino-acid residue, and X₈ is an amino acidselected from among alanine, leucine, methionine, asparagine, cysteine,aspartic acid, glutamic acid, phenylalanine, glycine, histidine, lysine,proline, arginine, serine, threonine, tyrosine and tryptophan.

According to another embodiment, in the improved nitrile hydratase thathas the amino-acid sequence shown in SEQ ID NO: 120, X₁ is M(methionine), X₂ is A (alanine), X₃ is S (serine), X₄ is L (leucine), X₅is Y (tyrosine), X₆ is A (alanine), X₇ is E (glutamic acid), and X₈ isan amino acid selected from among alanine, leucine, methionine,asparagine, cysteine, aspartic acid, glutamic acid, phenylalanine,glycine, histidine, lysine, proline, arginine, serine, threonine,tyrosine and tryptophan.

Another example of the improved nitrile hydratase of the presentinvention is as follows: in the amino-acid sequence of a known nitrilehydratase identified as SEQ ID NO: 4, the amino-acid residue at position83 (glutamine) of the α subunit is substituted with alanine, leucine,methionine, asparagine, cysteine, aspartic acid, glutamic acid,phenylalanine, glycine, histidine, lysine, proline, arginine, serine,threonine, tyrosine or tryptophan.

Modes of such amino-acid substitutions are denoted, for example, asQα83A, Qα83C, Qα83D, Qα83E, Qα83F, Qα83G, Qα83H, Qα83K, Qα83L, Qα83M,Qα83N, Qα83P, Qα83R, Qα83S, Qα83T, Qα83Y and Qα83W. Amino acids areidentified by a single-letter alphabetic code. The letter to the left ofthe numeral showing the number of amino-acid residues counted from theterminal to the substituted position (for example, “83”) represents theamino acid in a one-letter code before substitution, and the letter tothe right represents the amino acid in a one-letter code aftersubstitution.

In particular, when the amino-acid sequence of the α subunit in SEQ IDNO: 4 is denoted as “Qα83A” in the improved nitrile hydratase, theabbreviated notation means that, in the amino-acid sequence of the αsubunit (SEQ ID NO: 4), glutamine (Q) at position 83 counted from theN-terminal amino-acid residue (including the N-terminal amino-acidresidue itself) is substituted with alanine (A).

Modes of amino-acid substitutions in more preferred embodiments of theimproved nitrile hydratase according to the present invention are shownas the following 1˜17:

1. Qα83A,

2. Qα83C,

3. Qα83D,

4. Qα83E,

5. Qα83F,

6. Qα83G,

7. Qα83H,

8. Qα83K,

9. Qα83L,

10. Qα83M,

11. Qα83N,

12. Qα83P,

13. Qα83R,

14. Qα83S,

15. Qα83T,

16. Qα83Y and

17. Qα83W.

Preferred embodiments of base substitutions to cause the aboveamino-acid substitutions are shown below.

TABLE 2 amino-acid substitution base substitution Qα83A Base sequenceCAG (positions at 247~249 in SEQ ID NO: 3) is preferred to besubstituted with GCA, GCC, GCG, or GCT. Especially preferred to besubstituted is C at position 247 with G, A at position 248 with C, and Gat position 249 with C (CAG→GCC). Qα83C Base sequence CAG (positions at247~249 in SEQ ID NO: 3) is preferred to be substituted with TGC or TGT.Especially preferred to be substituted is C at position 247 with T, A atposition 248 with G, and G at position 249 with C (CAG→TGC). Qα83D Basesequence CAG (positions at 247~249 in SEQ ID NO: 3) is preferred to besubstituted with GAC or GAT. Especially preferred to be substituted is Cat position 247 with G, and G at position 249 with C (CAG→GAC). Qα83EBase sequence CAG (positions at 247~249 in SEQ ID NO: 3) is preferred tobe substituted with GAG or GAA. Especially preferred to be substitutedis C at position 247 with G (CAG→GAG). Qα83F Base sequence CAG(positions at 247~249 in SEQ ID NO: 3) is preferred to be substitutedwith TTC or TTT. Especially preferred to be substituted is C at position247 with T, A at position 248 with T, and G at position 249 with C(CAG→TTC). Qα83G Base sequence CAG (positions at 247~249 in SEQ ID NO:3) is preferred to be substituted with GGA, GGC, GGG or GGT Especiallypreferred to be substituted is C at position 247 with G, A at position248 with G, and G at position 249 with C (CAG→GGC). Qα83H Base sequenceCAG (positions at 247~249 in SEQ ID NO: 3) is preferred to besubstituted with CAC or CAT. Especially preferred to be substituted is Gat position 249 with C (CAG→CAC). Qα83K Base sequence CAG (positions at247~249 in SEQ ID NO: 3) is preferred to be substituted with AAA or AAG.Especially preferred to be substituted is C at position 247 with A(CAG→AAG). Qα83L Base sequence CAG (positions at 247~249 in SEQ ID NO:3) is preferred to be substituted with CTA, CTC, CTG, CTT, TTA or TTG.Especially preferred to be substituted is A at position 248 with T, andG at position 249 with C (CAG→CTC). Qα83M Base sequence CAG (positionsat 247~249 in SEQ ID NO: 3) is preferred to be substituted with ATG.Especially preferred to be substituted is C at position 247 with A, andA at position 248 with T (CAG→ATG). Qα83N Base sequence CAG (positionsat 247~249 in SEQ ID NO: 3) is preferred to be substituted with AAC orAAT. Especially preferred to be substituted is C at position 247 with A,and G at position 249 with C (CAG→AAC). Qα83P Base sequence CAG(positions at 247~249 in SEQ ID NO: 3) is preferred to be substitutedwith CCA, CCC, CCG or CCT. Especially preferred to be substituted is Aat position 248 with C (CAG→CCG). Qα83R Base sequence CAG (positions at247~249 in SEQ ID NO: 3) is preferred to be substituted with CGA, CGC,CGG, CGT, AGA or AGG. Especially preferred to be substituted is A atposition 248 with G (CAG→CGG). Qα83S Base sequence CAG (positions at247~249 in SEQ ID NO: 3) is preferred to be substituted with TCA, TCC,TCG, TCT, AGC or AGT. Especially preferred to be substituted is C atposition 247 with T, A at position 248 with C, and G at position 249with C (CAG→TCC). Qα83T Base sequence CAG (positions at 247~249 in SEQID NO: 3) is preferred to be substituted with ACA, ACC, ACG or ACT.Especially preferred to be substituted is C at position 247 with A, A atposition 248 with C, and G at position 249 with C (CAG→ACC). Qα83Y Basesequence CAG (positions at 247~249 in SEQ ID NO: 3) is preferred to besubstituted with TAC or TAT. Especially preferred to be substituted is Cat position 247 with T, and G at position 249 with C (CAG→TAC). Qα83WBase sequence CAG (positions at 247~249 in SEQ ID NO: 3) is preferred tobe substituted with TGG. Especially preferred to be substituted is C atposition 247 with T, and A at position 248 with G (CAG→TGG).(b-4) Improved Nitrile Hydratase (α82)

FIGS. 10-1 and 10-2 show amino-acid sequence alignments (in theone-letter code) in α-subunits of known nitrile hydratases derived fromvarious microorganisms. FIGS. 10-1 and 10-2 each show amino-acidsequences in sequence ID numbers 4, 105˜108, 121, 109, 110, 112, 111,122˜124, 113, 114, 125 from the top.

Furthermore, the improved nitrile hydratase of the present inventionincludes examples in which one or more (for example, 1˜10, preferred tobe approximately 1˜5) amino-acid residues are deleted, substitutedand/or added in the amino-acid sequences of known nitrile hydratases,excluding the amino-acid sequence identified as SEQ ID NO: 131. Examplesof the improved nitrile hydratase are described in patent publications5˜9 (the contents are incorporated by reference into the presentapplication). Nitrile hydratases in patent publication 5˜9 each exhibitheat resistance and acrylamide resistance. Moreover, as a result ofamino-acid substitutions of the present invention, enhanced catalyticactivity is further added to their properties.

An example of the improved nitrile hydratase of the present inventionhas an amino-acid sequence as shown in SEQ ID NO: 131 in the α subunitas shown in FIG. 11. Here, an amino-acid sequence shown in SEQ ID NO:132 is located at positions 73˜83 counted from the N-terminal.

According to an embodiment of the present invention, in the improvednitrile hydratase that has the amino-acid sequence shown in SEQ ID NO:131, X₁˜X₆ each independently indicate any amino-acid residue, and X₇ isan amino acid selected from among cysteine, phenylalanine, histidine,isoleucine, lysine, methionine, glutamine, arginine, threonine andtyrosine.

According to another embodiment, in the improved nitrile hydratase thathas the amino-acid sequence shown in SEQ ID NO: 131, X₁ is M(methionine), X₂ is A (alanine), X₃ is S (serine), X₄ is L (leucine), X₅is Y (tyrosine), X₆ is A (alanine), and X₇ is an amino acid selectedfrom among cysteine, phenylalanine, histidine, isoleucine, lysine,methionine, glutamine, arginine, threonine and tyrosine.

Another example of the improved nitrile hydratase of the presentinvention is as follows: in the amino-acid sequence of a known nitrilehydratase shown in SEQ ID NO: 4, the amino-acid residue at position 82(glutamic acid) of the α subunit is substituted with cysteine,phenylalanine, histidine, isoleucine, lysine, methionine, glutamine,arginine, threonine or tyrosine.

Modes of such amino-acid substitutions are denoted, for example, asEα82C, Eα82F, Eα82H, Eα82I, Eα82K, Eα82M, Eα82Q, Eα82R, Eα82T and Eα82Y.Amino acids are identified by a single-letter alphabetic code. Theletter to the left of the numeral showing the number of amino-acidresidues counted from the terminal to the substituted position (forexample, “82”) is the amino acid in a one-letter code beforesubstitution, and the letter to the right represents the amino acid in aone-letter code after substitution.

In particular, when the amino-acid sequence of the α subunit in SEQ IDNO: 4 is denoted as “Eα82C” in the improved nitrile hydratase, theabbreviated notation means among the amino-acid sequence of the αsubunit, glutamic acid (E) at position 82 counted from the N-terminalamino-acid residue (including the N-terminal amino-acid residue itself)is substituted with cysteine (C).

Modes of amino acid substitutions in more preferred embodiments of theimproved nitrile hydratase according to the present invention are shownas the following 1˜10:

1. Eα82C,

2. Eα82F,

3. Eα82H,

4. Eα82I,

5. Eα82K,

6. Eα82M,

7. Eα82Q,

8. Eα82R,

9. Eα82T and

10. Eα82Y.

Preferred embodiments of base substitutions to cause above amino-acidsubstitutions are shown below.

TABLE 3 amino-acid substitution base substitution Eα82C Base sequenceGAG (positions at 244~246 in SEQ ID NO: 3) is preferred to besubstituted with TGC or TGT. Especially preferred to be substituted is Gat position 244 with T, A at position 245 with G, and G at position 246with C (GAG→TGC). Eα82F Base sequence GAG (positions at 244~246 in SEQID NO: 3) is preferred to be substituted with TTC or TTT. Especiallypreferred to be substituted is G at position 244 with T, A at position245 with T, and G at position 246 with C (GAG→TTC). Eα82H Base sequenceGAG (positions at 244~246 in SEQ ID NO: 3) is preferred to besubstituted with CAT or CAC. Especially preferred to be substituted is Gat position 244 with C, and G at position 246 with C (GAG→CAC). Eα82IBase sequence GAG (positions at 244~246 in SEQ ID NO: 3) is preferred tobe substituted with ATT, ATC or ATA. Especially preferred to besubstituted is G at position 244 with A, A at position 245 with T, and Gat position 246 with C (GAG→ATC). Eα82K Base sequence GAG (positions at244~246 in SEQ ID NO: 3) is preferred to be substituted with AAA or AAG.Especially preferred to be substituted is G at position 244 with A(GAG→AAG). Eα82M Base sequence GAG (positions at 244~246 in SEQ ID NO:3) is preferred to be substituted with ATG. Especially preferred to besubstituted is G at position 244 with A, and A at position 245 with T(GAG→ATG). Eα82Q Base sequence GAG (positions at 244~246 in SEQ ID NO:3) is preferred to be substituted with CAA or CAG. Especially preferredto be substituted is G at position 244 with C (GAG→CAG). Eα82R Basesequence GAG (positions at 244~246 in SEQ ID NO: 3) is preferred to besubstituted with CGA, CGC, CGG, CGT, AGA or AGG. Especially preferred tobe substituted is G at position 244 with C, and A at position 245 with G(GAG→CGG). Eα82T Base sequence GAG (positions at 244~246 in SEQ ID NO:3) is preferred to be substituted with ACA, ACC, ACG or ACT. Especiallypreferred to be substituted is G at position 244 with A, A at position245 with C, and G at position 246 with C (GAG→ACC). Eα82Y Base sequenceGAG (positions at 244~246 in SEQ ID NO: 3) is preferred to besubstituted with TAT or TAC. Especially preferred to be substituted is Gat position 244 with T, and G at position 246 with G (GAG→TAC).(b-5) Improved Nitrile Hydratase (α85)

FIGS. 12-1 and 12-2 show the alignments of amino-acid sequences (in theone-letter code) in α-subunits of known nitrile hydratases derived fromvarious microorganisms. FIGS. 12-1 and 12-2 each show amino-acidsequences in sequence ID numbers 4, 105˜108, 121, 109, 110, 112, 111,122˜124, 113, 114, 125 from the top.

Furthermore, the improved nitrile hydratase of the present inventionincludes examples in which one or more (for example, 1˜10, preferred tobe approximately 1˜5) amino-acid residues are deleted, substitutedand/or added in the amino-acid sequences of known nitrile hydratases,excluding the amino-acid sequence identified as SEQ ID NO: 135. Examplesof such a nitrile hydratase are described in patent publications 5˜9(the contents are incorporated by reference into the presentapplication). Nitrile hydratases in patent publication 5˜9 each exhibitheat resistance and acrylamide resistance. Moreover, as a result ofamino-acid substitutions of the present invention, enhanced catalyticactivity is further added to their properties.

An example of the improved nitrile hydratase of the present inventionhas an amino-acid sequence as shown in SEQ ID NO: 135 in the α subunitas shown in FIG. 13. Here, an amino-acid sequence shown in SEQ ID NO:136 is located at positions 73˜85 counted from the N-terminal.

According to an embodiment of the present invention, in the improvednitrile hydratase that has the amino-acid sequence shown in SEQ ID NO:135, X₁˜X₈ each independently indicate any amino-acid residue, and X₉ isan amino acid selected from among cysteine, glutamic acid,phenylalanine, isoleucine, asparagine, glutamine, serine and tyrosine.

According to another embodiment, in the improved nitrile hydratase thathas the amino-acid sequence shown in SEQ ID NO: 135, X₁ is M(methionine), X₂ is A (alanine), X₃ is S (serine), X₄ is L (leucine), X₅is Y (tyrosine), X₆ is A (alanine), X₇ is E (glutamic acid), X₈ is A(alanine), and X₉ is an amino acid selected from among cysteine,glutamic acid, phenylalanine, isoleucine, asparagine, glutamine, serineand tyrosine.

Another example of the improved nitrile hydratase of the presentinvention is as follows: in the amino-acid sequence of a known nitrilehydratase shown in SEQ ID NO: 4, the amino-acid residue at position 85(histidine) of the α subunit is substituted with cysteine, glutamicacid, phenylalanine, isoleucine, asparagine, glutamine, serine ortyrosine.

Modes of such amino-acid substitutions are shown, for example, as Hα85C,Hα85E, Hα85F, Hα85I, Hα85N, Hα85Q, Hα85S and Hα85Y. Amino acids areidentified by a single-letter alphabetic code. The letter to the left ofthe numeral showing the number of amino-acid residues counted from theterminal to the substituted position (for example, “85”) is the aminoacid in a one-letter code before substitution, and the letter to theright represents the amino acid in a one-letter code after substitution.

In particular, when the amino-acid sequence of the α subunit in SEQ IDNO: 4 is denoted as “Hα85C” in the improved nitrile hydratase, theabbreviated notation means that, in the amino-acid sequence of the αsubunit (SEQ ID NO: 4), histidine (H) at position 85 counted from theN-terminal amino-acid residue (including the N-terminal amino-acidresidue itself) is substituted with cysteine (C).

Modes of amino acid substitutions in more preferred embodiments of theimproved nitrile hydratase according to the present invention are shownas the following 1˜8:

1. Hα85C,

2. Hα85E,

3. Hα85F,

4. Hα85I,

5. Hα85N,

6. Hα85Q,

7. Hα85S and

8. Hα85Y.

Preferred embodiments of base substitutions to cause the aboveamino-acid substitutions are shown below.

TABLE 4 amino-acid substitution base substitution Hα85C Base sequenceCAC (positions at 253~255 in SEQ ID NO: 3) is preferred to besubstituted with TGC or TGT. Especially preferred to be substituted is Cat position 253 with T, and A at position 254 with G (CAC→TGC). Hα85EBase sequence CAC (positions at 253~255 in SEQ ID NO: 3) is preferred tobe substituted with GAG or GAA. Especially preferred to be substitutedis C at position 253 with G, and C at position 255 with G (CAC→GAG).Hα85F Base sequence CAC (positions at 253~255 in SEQ ID NO: 3) ispreferred to be substituted with TTC or TTT. Especially preferred to besubstituted is C at position 253 with T, and A at position 254 with T(CAC→TTC). Hα85I Base sequence CAC (positions at 253~255 in SEQ ID NO:3) is preferred to be substituted with ATT, ATC or ATA. Especiallypreferred to be substituted is C at position 253 with A, and A atposition 254 with T (CAC→ATC). Hα85N Base sequence CAC (positions at253~255 in SEQ ID NO: 3) is preferred to be substituted with AAC or AAT.Especially preferred to be substituted is C at position 253 with A(CAC→AAC). Hα85Q Base sequence CAC (positions at 253~255 in SEQ ID NO:3) is preferred to be substituted with CAA or CAG. Especially preferredto be substituted is C at position 255 with G (CAC→CAG). Hα85S Basesequence CAC (positions at 253~255 in SEQ ID NO: 3) is preferred to besubstituted with TCA, TCC, TCG, TCT, AGC or AGT. Especially preferred tobe substituted is C at position 253 with T, and A at position 254 with C(CAC→TCC). Hα85Y Base sequence CAC (positions at 253~255 in SEQ ID NO:3) is preferred to be substituted with TAT or TAC. Especially preferredto be substituted is C at position 253 with T (CAC→TAC).(b-6) Nitrile Hydratase Activity

Among the activity properties of the improved nitrile hydrataseaccording to the present invention, catalytic activity is improvedrelative to that in a nitrile hydratase before a mutation is introduced.

Here, “nitrile hydratase activity” means an enzyme to catalyze thehydration for converting a nitrile compound to a corresponding amidecompound (RCN+H₂O→RCONH₂). Determining the activity is conducted bybringing a nitrile compound as a substrate into contact with a nitrilehydratase for conversion to a corresponding amide compound and bydetermining the resultant amide compound. Any nitrile compound may beused as a substrate as long as nitrile hydratase reacts with such acompound, but acrylonitrile is preferred.

Reaction conditions are a substrate concentration of 2.5%, reactiontemperature of 10° C. to 30° C. and duration of 10˜30 minutes. Theenzymatic reactions are terminated by adding phosphoric acid. Then,using HPLC (high-performance liquid chromatography) or gaschromatography, the produced acrylamide is analyzed to measure theamount of the amide compound.

“Improved catalytic activity” means that when activity is measured inthe culture of a transformant containing the improved nitrile hydrataseor the improved nitrile hydratase isolated from the transformant, thecatalytic activity of the improved nitrile hydratase is at least 10%higher than that of the parent strain measured under the sameconditions. The parent strain in the present application means atransformant into which a template plasmid for mutation was introduced.

As for an amide compound, an amide compound represented by the generalformula (1) below, for example, is preferred.R—CONH₂  (1)(Here, R is an optionally substituted linear or branched alkyl oralkenyl group having 1˜10 carbon atoms, an optionally substitutedcycloalkyl or allyl group having 3˜18 carbon atoms, or an optionallysubstituted saturated or unsaturated heterocyclic group.) Especiallypreferred is an acrylamide in which “R” in the formula is “CH₂═CH—.”

The above improved nitrile hydratase is obtained by performingamino-acid substitution on a nitrile hydratase. For example, such animproved nitrile hydratase is obtained by modifying the amino-acidsequence (SEQ ID NO: 2) of a nitrile hydratase derived from Rhodococcusrhodocrous J1 strain, and by screening a nitrile hydratase with animproved catalytic activity.

Rhodococcus rhodochrous J1 strain is internationally registered underaccession number “FERM BP-1478” at the International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki), deposited Sep.18, 1987.

Using a nitrile hydratase derived from bacteria other than the J1strain, catalytic activity is thought to be improved as well when amutation is introduced by modifying a position, type of amino acid orDNA sequence described above. Preferred strains are: Rhodococcusrhodocrous M8 (SU 1731814) (SEQ ID NO: 5), Rhodococcus ruber TH (SEQ IDNO: 6), Rhodococcus rhodocrous M33 (VKM Ac-1515D), Rhodococcuspyridinivorans MW3 (SEQ ID NO: 7), Rhodococcus pyridinivorans S85-2 (SEQID NO: 8), Rhodococcus pyridinivorans MS-38 (SEQ ID NO: 9), Rhodococcusruber RH (CN 101463358) (SEQ ID NO: 52), Nocardia sp. JBRs (SEQ ID NO:10), Nocardia sp. YS-2002 (SEQ ID NO: 11), Rhodococcus rhodocrous ATCC39384 (SEQ ID NO: 12), uncultured bacterium SP1 (SEQ ID NO: 42),uncultured bacterium BD2 (SEQ ID NO: 43), Comamonas testosterone (SEQ IDNO: 44), Geobacillus thermoglucosidasius Q6 (SEQ ID NO: 45),Pseudonocardia thermophila JCM 3095 (SEQ ID NO: 46), Rhodococcusrhodocrous Cr 4 (SEQ ID NO: 47), or the like. Obtained through naturalmutation from the M8 strain above (SU 1731814), Rhodococcus rhodocrousM33 (VKM Ac-1515D) was selected because it is capable of constitutiveexpression of a nitrile hydratase. The amino-acid or gene sequence ofthe nitrile hydratase itself is not mutated (U.S. Pat. No. 5,827,699).In the β subunit in a bacterium listed above, the amino-acid residue atposition 48 from the N-terminal of the improved nitrile hydratase issubstituted with cysteine, aspartic acid, glutamic acid, histidine,isoleucine, lysine, methionine, asparagine, proline, glutamine, serineor threonine.

Methods for conducting amino-acid substitution on a wild-type nitrilehydratase are as follows: a bacterium having nitrile hydratase activityis brought into contact for reactions with chemicals such as hydroxylamine or nitrous acid as a mutation source; UV rays are irradiated toinduce mutation; error-prone PCR or site-directed mutagenesis isemployed to introduce a mutation at random into the gene that encodes anitrile hydratase; and the like.

(b-7) Error-Prone PCR

To study functions and characteristics of proteins using a mutant,random mutagenesis is known. Random mutagenesis is a method to introducea random mutation to the gene encoding a specific protein so that amutant is produced. In random mutagenesis by PCR, stringency conditionsare set low for the DNA amplification period so that a mutant base isintroduced (error-prone PCR).

In such an error-prone PCR method, a mutation is introduced randomlyinto any position of the entire DNA site to be amplified. Then, byexamining the function of the obtained mutant, which occurred throughthe mutation introduced at a random site, information of the amino acidor domain important for a specific function of a protein is obtained.

As a nitrile hydratase used for the template of error-prone PCR, thenitrile hydratase gene derived from a wild-type strain or DNA obtainedas an amplified product by error-prone PCR is used.

As reaction conditions for error-prone PCR, for example, a compositionratio of any one, two or three among dNTP (dGTP, dCTP, dATP or dTTP) inthe reaction mix is reduced relative to another dNTP. In so setting,during the DNA synthesis, at a position that requires a dNTP whose ratiois reduced, another dNTP is more likely to be used by error and that maylead to mutation. In addition, other preferred reaction conditions are acomposition in which the amount of MgCl₂ and/or MnCl₂ in the reactionmix is increased.

(b-8) Improved Nitrile Hydratase Mutagenesis

Based on a known nitrile hydratase gene, DNA that encodes such animproved nitrile hydtratase is produced by site-directed mutagenesismethods described in Molecular Cloning, A Laboratory Manual, 2ndedition, Cold Spring Harbor Laboratory Press (1989), Current Protocolsin Molecular Biology, John Wiley and Sons (1987-1997) and the like. Tointroduce a mutation into DNA by well-known methods such as the Kunkelmethod or Gapped Duplex method, mutagenesis kits applying site-directedmutagenesis methods such as follows are used: QuickChange™ XLSite-Directed Mutagenesis Kit (made by Stratagene), GeneTailor™Site-Directed Mutagenesis System (made by Invitrogen Corporation),TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Kmand the like, made by Takara Bio Inc.) and the like.

Furthermore, the DNA related to the present invention includes DNA whichis hybridized under stringent conditions with a DNA made up of a basesequence complementary to the base sequence of the DNA of the presentinvention, and which encodes a protein having nitrile hydrataseactivity.

Such an improved nitrile hydratase DNA is obtained by introducing amutation into a wild-type gene as described above. Alternatively, usingthe DNA sequence or its complementary sequence or a DNA fragment as aprobe, improved nitrile hydratase DNA may also be obtained from cDNAlibraries and genomic libraries by employing well-known hybridizationmethods such as colony hybridization, plaque hybridization, Southernblot or the like. Libraries constructed by a well-known method may beused, or commercially available cDNA libraries and genomic libraries mayalso be used.

“Stringent conditions” are those for washing after hybridization; a saltconcentration of 300˜2000 mM and a temperature of 40˜75° C., preferablya salt concentration of 600˜900 mM and a temperature of 65° C. Forexample, conditions 2×SSC at 50° C. may be employed. In addition to sucha salt concentration of the buffer, temperature and the like, a personskilled in the art may set conditions for obtaining DNA that encodes anitrile hydratase of the present invention by adding various conditionssuch as probe concentration, probe length and reaction time.

For detailed procedures for hybridization, Molecular Cloning, ALaboratory Manual, 2nd edition (Cold Spring Harbor Laboratory Press(1989)) or the like may be referred to. DNA to be hybridized includesDNA or its fragment, containing a base sequence which is at least 40%,preferably 60%, more preferably 90% or greater, homologous to thegenomic DNA of the present invention.

(c) Recombinant Vector, Transformant

It is necessary for a nitrile hydratase gene to be put into a vector sothat nitrile hydratase is expressed in the host organism to betransformed. Examples of such vectors are plasmid DNA, bacteriophageDNA, retrotransposon DNA, artificial chromosome DNA and the like.

In addition, a host to be used in the present invention is not limitedto any specific type as long as it can express the target nitrilehydratase after the recombinant vector is introduced into the host.Examples are bacteria such as E. coli and Bacillus subtilis, yeasts,animal cells, insect cells, plant cells and the like. When E. coli isused as a host, an expression vector with high expression efficiency,such as expression vector pkk 233-2 with a trc promoter (made byAmersham Biosciences Corp.), pTrc 99A (made by Amersham BiosciencesCorp.) or the like, is preferred.

In addition to a nitrile hydratase gene, a vector may be coupled with apromoter, terminator, enhancer, splicing signal, poly A addition signal,selection marker, ribosome binding sequence (SD sequence) or the like.Examples of selection markers are kanamycin resistance gene,dihydrofolate reductase gene, ampicillin resistance gene, neomycinresistance gene and the like.

When a bacterium is used as a host, Escherichia coli may be used, forexample, and a Rhodococcus strain such as Rhodococcus rhodochrous ATCC12674, Rhodococcus rhodochrous ATCC 17895 and Rhodococcus rhodochrousATCC 19140 may also be used. Those ATCC strains are obtained from theAmerican type culture collection.

When E. coli is used as a host for producing a transformant to express anitrile hydratase, since most of the expressed nitrile hydratase isformed as an inclusion body and is insoluble, a transformant with lowcatalytic activity is obtained. On the other hand, if a Rhodococcusstrain is used as a host, nitrile hydratase is present in the solublefraction, and a transformant with high activity is obtained. Thosetransformants may be selected based on purposes. However, when animproved enzyme is selected under stringent conditions, a transformantwith high activity derived from a Rhodococcus strain is preferred.

Introducing a recombinant vector into a bacterium is not limited to anyspecific method as long as DNA is introduced into the bacterium. Forexample, a method using calcium ions, electroporation or the like may beemployed.

When yeast is used as a host, examples are Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pichia pastoris and the like. As a method forintroducing a recombinant vector into yeast, it is not limitedspecifically as long as DNA is introduced into the yeast. For example,an electroporation method, spheroplast method, lithium acetate method orthe like may be employed.

When animal cells are used as a host, monkey cells COS-7, Vero, CHOcells, mouse L cells, rat GH3 cells, human FL cells or the like may beemployed. As a method for introducing a recombinant vector into animalcells, for example, an electroporation method, calcium phosphate method,lipofection method or the like may be used.

When insect cells are used as a host, Sf9 cells, Sf21 cells or the likemay be used. A method for introducing a recombinant vector into insectcells, for example, a calcium phosphate method, lipofection method,electroporation method or the like may be used.

When plant cells are used as a host, tobacco BY-2 cells or the like maybe used. However, that is not the only option. A method for introducinga recombinant vector into plant cells, for example, an Agrobacteriummethod, particle gun method, PEG method, electroporation method or thelike may be used.

(d) Method for Producing Culture and Improved Nitrile Hydratase

An improved nitrile hydratase of the present invention is obtained byincubating the above transformant and by collecting from the obtainedculture.

The present invention also relates to a method for producing an improvednitrile hydratase, and the method is characterized by collecting animproved nitrile hydratase from the culture above.

In the present invention, “culture” means any of culture supernatant,cell cultured cell, bacterial-cell culture, and cell homogenates orbacterial-cell homogenates. To incubate a transformant of the presentinvention, a generally used method for incubating a host is used. Thetarget nitrile hydratase is accumulated in the culture.

As for a culture to incubate a transformant of the present invention, anatural or synthetic culture medium is used as long as it contains acarbon source, a nitrogen source, inorganic salts or the like for thehost bacteria to assimilate, and incubation of a transformant isperformed efficiently. Examples of a carbon source are carbohydratessuch as glucose, galactose, fructose, sucrose, raffinose and starch;organic acids such as acetic acid and propionic acid; alcohols such asethanol and propanol; and the like. Examples of a nitrogen source areinorganic acids such as ammonia, ammonium chloride, ammonium sulfate,ammonium acetate and ammonium phosphate; ammonium salts of organicacids; and other nitrogen-containing compounds.

In addition, peptone, yeast extract, meat extract, corn steep liquor,various amino acids or the like may also be used. Examples of mineralsare monopotassium phosphate, potassium dihydrogenphosphate, magnesiumphosphate, magnesium sulfate, sodium chloride, ferrous sulfate,manganese sulfate, zinc sulfate, copper sulfate, calcium carbonate andthe like. Also, if necessary, a defoaming agent may be used to preventfoaming during the incubation process. Moreover, cobalt ions or ironions as prosthetic molecules of a nitrile hydratase, or nitriles andamides as an inducer of the enzyme, may also be added to the culture.

Incubation may be conducted by adding selective pressure to prevent thevector and the target gene from being eliminated. Namely, if a selectionmarker is a drug-resistant gene, a corresponding chemical agent may beadded; or if a selection marker is an auxotrophic complementary gene,corresponding nutrition factors may be removed.

Also, if a selection marker has a genetic assimilation trait, anequivalent assimilation factor may be added as a sole factor ifnecessary. For example, when E. coli transformed by a vector containingan ampicillin-resistant gene is incubated, ampicillin may be added asneeded during the incubation process.

When incubating a transformant transformed by an expression vectorcontaining an inducible promoter, such an inducer may be added to theculture if necessary. For example, when incubating a transformanttransformed by an expression vector with a promoter inducible withisopropyl-β-D-thiogalactopyranoside (IPTG), IPTG or the like may beadded to the culture. Likewise, when incubating a transformanttransformed by an expression vector with a trp promoter inducible withindoleacetic acid (IAA), IAA or the like may be added to the culture.

Incubation conditions of a transformant are not limited specifically aslong as the productivity of the target nitrile hydratase and growth ofthe host are not prohibited. Generally, conditions are preferred to be10° C.˜40° C., more preferably 20° C.˜37° C., for 5˜100 hours. The pHvalue is adjusted using inorganic or organic acid, alkaline solution orthe like. If it is an E. coli, the pH is adjusted to be 6˜9.

As for incubation methods, solid-state culture, static culture, shakingculture, aeration-agitation culture and the like may be used. When an E.coli transformant is incubated, it is especially preferred to useshaking culture or aeration-agitation culture (jar fermentation) underaerobic conditions.

When incubated in culture conditions above, the improved nitrilehydratase of the present invention is accumulated at a high yield in theabove culture medium, namely, at least in any of culture supernatant,cell culture, bacterial-cell culture, cell homogenates or bacterial-cellhomogenates.

When an improved nitrile hydratase is incubated and produced in a cellor bacterial cell, the target nitrile hydratase is collected byhomogenizing the cells or bacterial cells. Cells or bacterial cells arehomogenized by high-pressure treatment using a French press orhomogenizer, supersonic treatment, grinding treatment using glass beadsor the like, enzyme treatment using lysozyme, cellulose, pectinase andthe like, freezing and thawing treatment, hypotonic solution treatment,bacteriolysis induction treatment by phage, and so on.

After the homogenization process, residues of cell homogenates orbacterial-cell homogenates (including insoluble fractions of the cellextract) are removed if necessary. To remove residues, centrifugal orfiltration methods are employed. To increase the efficiency of removingresidues, a coagulant or filter aid may be used. The supernatantobtained after the removal of residues is soluble fractions of the cellextract, which are used as a crudely purified improved nitrile hydratasesolution.

Also, when an improved nitrile hydratase is produced in a bacterial cellor in cells, it is an option to collect the bacterial cell or the cellsthemselves by a centrifuge or membrane filtration and to use withouthomogenizing them.

When an improved nitrile hydratase is produced outside cells orbacterial cells, the culture may be used as is, or the cells orbacterial cells are removed using a centrifugal or filtration method.Then, the improved nitrile hydratase is collected from the culture bybeing extracted through ammonium sulfate precipitation, if necessary.Furthermore, dialysis or various chromatography techniques (gelfiltration, ion exchange chromatography, affinity chromatography, etc.)may be used to isolate and purify the nitrile hydratase.

To check the production yield of a nitrile hydratase obtained byincubating a transformant is not limited to using any specific method,but SDS-PAGE (polyacrylamide gel electrophoresis), nitrile hydrataseactivity measurements or the like may be used to determine the yield perculture, per wet or dry weight in a bacterial cell, or per crudeenzymatic protein. SDS-PAGE may be conducted by a method well known by aperson skilled in the art. Also, the activity described above may beapplied to nitrile hydratase activity.

Without using any living cells, an improved nitrile hydratase of thepresent invention may be produced using a cell-free protein synthesissystem.

In a cell-free protein synthesis system, a protein is produced in anartificial vessel such as a test tube using a cell extract. A cell-freeprotein synthesis system used in the present application includes acell-free transcription system that synthesizes RNA using DNA as atemplate.

In such a case, an organism corresponding to the above host is theorganism from which the cell extract is derived. Here, for the cellextract, extracts of eukaryotic or prokaryotic origin, such as theextract from wheat germ, E. coli and the like, may be used. Such cellextracts may be concentrated or not.

The cell extract is obtained by ultrafiltration, dialysis, polyethyleneglycol (PEG) precipitation or the like. In the present invention, acommercially available kit may also be used for cell-free proteinsynthesis. Examples of such a kit are a reagent kit PROTEIOS™ (Toyobo),TNT™ system (Promega KK), a synthesizer PG-Mate™ (Toyobo), RTS (RocheDiagnostics) and the like.

An improved nitrile hydratase obtained by cell-free protein synthesis asdescribed above is also purified by properly selecting a chromatographytype.

2. Method for Producing Amide Compound

The improved nitrile hydratase obtained above is used as an enzymaticcatalyst for material production. For example, an amide compound isproduced by bringing a nitrile compound into contact with the improvednitrile hydratase. Then, the amide compound produced upon contact iscollected. Accordingly, an amide compound is produced.

The isolated and purified nitrile hydratase as described above is usedas an enzymatic catalyst. In addition, a gene is introduced so as toexpress an improved nitrile hydratase in a proper host as describedabove and the culture after the host is incubated or the processedproducts of the culture may also be used. Processed products are, forexample, incubated cells immobilized with acrylamide gel or the like,those processed by glutaraldehyde, those supported by inorganic carrierssuch as alumina, silica, zeolite, diatomaceous earth and the like.

Here, “contact” means that an improved nitrile hydratase and a nitrilecompound are present in the same reaction system or incubation system:for example, an isolated and purified improved nitrile hydratase and anitrile compound are mixed; a nitrile compound is added into aincubation vessel of a cell to express an improved nitrile hydratasegene; cells are incubated in the presence of a nitrile compound; a cellextract is mixed with a nitrile compound; and so on.

A nitrile compound as a substrate is selected by considering thesubstrate specificity of the enzyme, stability of the enzyme in thesubstrate and the like. As for a nitrile compound, acrylonitrile ispreferred. The reaction method and the method for collecting an amidecompound after the completion of reactions are properly selecteddepending on the characteristics of the substrate and the enzymaticcatalyst.

The enzymatic catalyst is preferred to be recycled as long as itsactivity is not deactivated. From the viewpoint of preventingdeactivation and of recycling ease, the enzymatic catalyst is preferredto be used as a processed product.

EXAMPLES

In the following, examples of the present invention are described indetail. However, the present invention is not limited to those.Rhodococcus rhodocrous J1 strain is registered under accession number“FERM BP-1478” at the International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (Central 6,1-1-1 Higashi, Tsukuba, Ibaraki), deposited Sep. 18, 1987.

Preparation Example 1

Preparation of Plasmid pSJ034

As a template to perform the amino-acid substitution of the presentinvention, plasmid pSJ034 (FIG. 1) having the nitrile hydratase gene ofthe J1 strain was produced by the following method.

Plasmid pSJ034 is capable of expressing nitrile hydratase in aRhodococcus strain. Plasmid pSJ034 was produced from pSJ023 by themethod disclosed in JP publication H10-337185. Namely, partially cleavedat the XbaI site and ligated with the Sse8387I linker, plasmid pSJ033was prepared so that one XbaI site of plasmid pSJ023 was substitutedwith Sse8387I. Next, plasmid pSJ033 was partially cleaved at theSse8387I site, and a Klenow fragment was used to blunt the ends so as tocause self ligation. Accordingly, plasmid pSJ034 was obtained. Here,pSJ023 is a transformant “R. rhodochrous ATCC 12674/pSJ023,” and isinternationally registered under accession number “FERM BP-6232” at theInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology (Central 6, 1-1-1 Higashi, Tsukuba,Ibaraki), deposited Mar. 4, 1997.

Preparation Example 2

Preparation of Plasmid pFR005

(1) Construction of Mutant Gene Library

As for a template plasmid, pER855A (FIG. 5) was used, prepared bymodifying plasmid pER855 (see JP publication 2010-172295) as follows:counted downstream from the N-terminal amino-acid residue of theamino-acid sequence (SEQ ID NO: 2) in the β subunit, an amino-acidresidue at position 167 was mutated from asparagine (N) to serine (S);an amino-acid residue at position 219 was mutated from valine (V) toalanine (A); an amino-acid residue at position 57 was mutated fromserine (S) to methionine (M); an amino-acid residue at position 114 wasmutated from lysine (K) to tyrosine (Y); and an amino-acid residue atposition 107 was mutated from threonine (T) to lysine (K).

First, introduction of a mutation into the nitrile hydratase gene wasconducted as follows:

<Composition of PCR Reaction Mixture>

sterile water 20 μL pER855A (1 ng/mL) 1 μL Forward primer (10 mM) 2 μLReverse primer (10 mM) 2 μL PrimeSTAR MAX (2×) 25 μL total 50 μL<PCR Reaction Conditions>(98° C. for 10 sec, 55° C. for 5 sec, 72° C. for 90 sec)×30 cycles<Primers> Primers for Saturation Mutagenesis at β17

β17RM-F: (SEQ ID NO: 63) ggatacggaccggtcNNStatcagaaggacgag β17RM-R:(SEQ ID NO: 64) ctcgtccttctgataSNNgaccggtccgtatcc<Reaction Conditions>(94° C. for 30 sec, 65° C. for 30 sec, 72° C. for 3 min)×30 cycles

After the completion of PCR, 5 μL of the reaction mixture was providedfor 0.7% agarose gel electrophoresis, an amplified fragment of 11 kb wasconfirmed, and 1 μL DpnI (provided with the kit) was added to the PCRreaction mixture, which was then reacted at 37° C. for an hour.Accordingly, the template plasmid was removed. After that, the reactionmixture was purified using Wizard SV Gel and PCR Clean-Up System(Promega KK), and transformation was introduced into JM109 using thepurified PCR reaction product. A few thousand obtained colonies werecollected from the plate, and plasmid DNA was extracted using QIAprepSpin Miniprep Kit (Qiagen) to construct a mutant gene library.

(2) Producing Rhodococcus Transformant

The cells of Rhodococcus rhodochrous strain ATCC 12674 at a logarithmicgrowth phase were collected by a centrifugal separator, washed withice-cooled sterile water three times and suspended in the sterile water.Then, 1 μL of plasmid prepared in (2) above and 10 μL of thebacterial-cell suspension were mixed and ice-cooled. The plasmid DNA andthe bacterial-cell suspension were supplied into a cuvette, and electricpulse treatment was conducted at 2.0 KV and 200Ω using anelectroporation device, Gene Pulser II (Bio-Rad Laboratories, Inc.).

The cuvette with the mixture processed by electric pulse was let standfor 10 minutes under ice-cold conditions, and a heat-shock treatment wasconducted at 37° C. for 10 minutes. Then, 500 μL of an MYK culturemedium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto maltextract, 0.2% K₂HPO₄, 0.2% KH₂PO₄) was added and let stand at 30° C. for5 hours, and the strain was then applied on an MYK agar mediumcontaining 50 μg/mL kanamycin. The colony obtained after being incubatedat 30° C. for 3 days was used as a transformant. In the same manner,transformant pER 855A was prepared as a comparative strain.

(3) Amide Treatment on Rhodococcus Strain Transformant

The Rhodococcus transformant containing nitrile hydratase gene, obtainedin (2) above and ATCC 12674/pER855A as a comparative strain were usedfor screening. In a 96-hole deep-well plate, 1 mL each of a GGPK culturemedium (1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05%K₂HPO₄, 0.05% KH₂PO₄, 0.05% MgSO₄.7H₂O, 1% CoCl₂, 0.1% urea, 50 μg/mLkanamycin, pH 7.2) was supplied. In each culture medium, the abovestrain was inoculated, and subjected to liquid culture at 30° C. for 3days.

Next, 30 μL of the liquid culture obtained above was dispensed in a96-hole plate and the culture medium was removed by centrifugation.Lastly, 40 μL of a 50% acrylamide solution was added to suspend thebacteria. The transformant suspended in a high-concentration acrylamidesolution was put in an incubator to completely deactivate thecomparative strain through heat treatment conducted at 50° C. for 30minutes. The remaining nitrile hydratase activity was measured asfollows.

First, after the acrylamide treatment, a transformant was washed with a50 mM phosphate buffer (pH 7.0) and the activity was measured by thefollowing method. The washed transformant and 50 mM phosphate buffer (pH7.0) were supplied to a test tube and preincubated at 30° C. for 10minutes, and an equivalent volume of a 5% acrylonitrile solution (pH7.0) was added and reacted for 10 minutes. Then, one tenth volume of 1 Mphosphoric acid was added to terminate the reaction. Next, thetransformant was removed from the terminated reaction mixture bycentrifugation, and the mixture was diluted to a proper concentrationfor analysis by HPLC (WAKOSIL 5C8 (Wako Pure Chemical Industries) 250 mmlong, 10% acetonitrile containing 5 mM phosphoric acid, flow rate ofmobile phase at 1 mL/min, wavelength of a UV absorption detector 260nm). Using untreated cells for which acrylamide treatment was notconducted, activity was measured for comparison. Then, based on theobtained activity values, the remaining activity after acrylamidetreatment was determined.

Among hundreds of transformants containing a mutant nitrile hydratasegene obtained above, mutant enzyme pFR005 showing resistance to ahigh-concentration acrylamide was selected.

(4) Confirming Base Sequence

To confirm the base sequence of the nitrile hydratase gene, plasmid wasrecovered from the selected strains. Rhodococcus transformants wereinoculated in 10 mL of an MYK culture medium (0.5% polypeptone, 0.3%Bacto yeast extract, 0.3% malt extract, 1% glucose, 50 μg/mL kanamycin)and incubated for 24 hours, and a 20% sterile glycine solution was addedto make the final concentration of 2%, and further incubated for another24 hours. Then, the bacterial cells were recovered by centrifugation,washed with a TES buffer (10 mM Tris-HCl (pH 8)-10 mM NaCl-1 mM EDTA),suspended in 2 mL of 50 mM Tris-HCl (pH8)-12.5% sucrose-100 mM NaCl-1mg/mL lysozyme, and subjected to shaking culture at 37° C. for 3 hours.Then, 0.4 mL of 10% SDS was added and the mixture was shaken gently foran hour at room temperature, to which 2.1 mL of 5 M sodium acetatebuffer (pH 5.2) was added and let stand in ice for an hour. Next, themixture was centrifuged for an hour at 10,000×g at 4° C. to obtain asupernatant, to which a 5-times volume ethanol was added and let standat −20° C. for 30 minutes. Then, the mixture was centrifuged at 10,000×gfor 20 minutes. The precipitate was washed with 10 mL of 70% ethanol anddissolved in 100 μL of a TE buffer. Accordingly, a DNA solution wasobtained.

Next, the sequence including nitrile hydratase was amplified by a PCRmethod.

<Composition of PCR Reaction Mixture>

template plasmid 1 μL 10× PCR buffer (made by NEB) 10 μL primer NH-19(50 μM) 1 μL primer NH-20 (50 μM) 1 μL 2.5 mM dNTPmix 8 μL sterile water79 μL Taq DNA polymerase (made by NEB) 1 μL<Primers>

NH-19: (SEQ ID NO: 65) GCCTCTAGATATCGCCATTCCGTTGCCGG NH-20:(SEQ ID NO: 66) ACCCTGCAGGCTCGGCGCACCGGATGCCCAC<Reaction Conditions>(94° C. for 30 sec, 65° C. for 30 sec, 72° C. for 3 min)×30 cycles

After completion of PCR, 5 μL of the reaction mixture was subjected to0.7% agarose gel electrophoresis to detect a 2.5 kb PCR amplifiedproduct. After Exo-SAP treatment (Amersham Pharmacia Biotech) on the PCRreaction mixture, samples for alignment analysis were prepared by acycle sequencing method, and were analyzed using CEQ-2000XL (BeckmanCoulter). As a result, the mutation positions of pFR005 were confirmedto be Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K and Pβ17G. Namely, inplasmid pFR005, proline at position 17 in the 1 subunit was mutated toglycine, serine at position 57 in the β subunit was mutated to lysine,tyrosine at position 107 in the β subunit was mutated to lysine, lysineat position 114 in the β subunit was mutated to tyrosine, asparagine atposition 167 in the β subunit was mutated to serine, and valine atposition 219 in the β subunit was mutated to alanine.

Example 1

Preparation of Improved Nitrile Hydratase

Using pSJ034 formed in preparation example 1, amino-acid substitutionwas conducted. The following composition of a reaction mixture, reactionconditions and primers were used for the PCR.

<Composition of PCR Reaction Mixture>

sterile water 20 μL pSJ034 (1 ng/mL) 1 μL Forward primer (10 mM) 2 μLReverse primer (10 mM) 2 μL PrimeSTAR MAX (2×) 25 μL total 50 μL<PCR Reaction Conditions>(98° C. for 10 sec, 55° C. for 5 sec, 72° C. for 90 sec)×30 cycles<Primers>

TABLE 5 substi- tuted SEQ amino  name of ID acid primer sequence NO Cβ48C-F TCGTGGTGCGACAAGTCGCGGTTCTTC 13 β48C-R CTTGTCGCACCACGATATGCCCTTGAG14 D β48D-F TCGTGGGACGACAAGTCGCGGTTCTTC 15 β48D-RCTTGTCGTCCCACGATATGCCCTTGAG 16 E β48E-F TCGTGGGAGGACAAGTCGCGGTTCTTC 17β48E-R CTTGTCCTCCCACGATATGCCCTTGAG 18 H β48H-FTCGTGGCACGACAAGTCGCGGTTCTTC 19 β48H-R CTTGTCGTGCCACGATATGCCCTTGAG 20 Iβ48I-F TCGTGGATCGACAAGTCGCGGTTCTTC 21 β48I-R CTTGTCGATCCACGATATGCCCTTGAG22 K β48K-F TCGTGGAAGGACAAGTCGCGGTTCTTC 23 β48K-RCTTGTCCTTCCACGATATGCCCTTGAG 24 M β48M-F TCGTGGATGGACAAGTCGCGGTTCTTC 25β48M-R CTTGTCCATCCACGATATGCCCTTGAG 26 N β48N-FTCGTGGAACGACAAGTCGCGGTTCTTC 27 β48N-R CTTGTCGTTCCACGATATGCCCTTGAG 28 Pβ48P-F TCGTGGCCGGACAAGTCGCGGTTCTTC 29 β48P-R CTTGTCCGGCCACGATATGCCCTTGAG30 Q β48Q-F TCGTGGCAGGACAAGTCGCGGTTCTTC 31 β48Q-RCTTGTCCTGCCACGATATGCCCTTGAG 32 S β48S-F TCGTGGTCCGACAAGTCGCGGTTCTTC 33β48S-R CTTGTCGGACCACGATATGCCCTTGAG 34 T β48T-FTCGTGGACCGACAAGTCGCGGTTCTTC 35 β48T-R CTTGTCGGTCCACGATATGCCCTTGAG 36

After the completion of PCR, 5 μL of the reaction mixture was subjectedto 0.7% agarose gel electrophoresis and an 11l-kb PCR amplified productwas detected. Then, 1 μL of DpnI (provided in the kit) was added to thePCR reaction mixture and reacted at 37° C. for an hour to remove thetemplate plasmid. After the reaction was completed, the reaction mixturewas purified using Wizard SV Gel and PCR Clean-Up System (made byPromega KK), and the purified PCR product was used to transform JM109.From the obtained culture, plasmid DNA was extracted using QIAprep SpinMiniprep Kit (made by Qiagen), and the base sequence of the nitrilehydratase was confirmed using automated sequencer CEQ 8000 (made byBeckman Coulter, Inc.). Obtained plasmids were named as follows.

TABLE 6 name of amino-acid plasmid substitution pSJ102 Wβ48C pSJ103Wβ48D pSJ104 Wβ48E pSJ107 Wβ48H pSJ108 Wβ48I pSJ109 Wβ48K pSJ111 Wβ48MpSJ112 Wβ48N pSJ113 Wβ48P pSJ114 Wβ48Q pSJ116 Wβ48S pSJ117 Wβ48T

Example 2

Preparation of Rhodococcus Transformant

Cells of Rhodococcus rhodocrous strain ATCC 12674 in a logarithmicgrowth phase were collected using a centrifuge, washed three times withice-cold sterile water, and suspended in the sterile water. Then, 1 μLof plasmid prepared in example 1 and 10 μL of the bacterial-cellsuspension were mixed and ice-cooled. The DNA and the bacterial-cellsuspension were supplied in a cuvette, and electric pulse treatment wasconducted using an electroporation device, Gene Pulser (Bio-RadLaboratories), under conditions of 2.0 kV and 200Ω. The electric-pulseprocessed mixture was let stand in an ice-cold condition for 10 minutes,and subjected to heat shock at 37° C. for 10 minutes. After 500 μL of anMYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3%Bacto malt extract, 0.2% K₂HPO₄, 0.2% KH₂PO₄) was added and let stand at30° C. for 5 hours, the strain was applied onto an MYK agar culturemedium containing 50 μg/mL kanamycin and incubated at 30° C. for 3 days.The obtained colony after incubating at 30° C. for 3 days was used as atransformant.

Each transformant obtained above was inoculated into an MYK culturemedium (50 μg/mL kanamycin), and subjected to shaking culture at 30° C.for 2 days. Then, 1% culture was inoculated into a GGPK culture medium(1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K₂HPO₄,0.05% KH₂PO₄, 0.05% Mg₂O₄.7H₂O, 1% CoCl₂, 0.1% urea, 50 μg/mL kanamycin,pH 7.2), and subjected to shaking culture at 30° C. for 3 days.Bacterial cells were collected by using a centrifuge, and were washedwith a 100 mM phosphate buffer (pH 7.0) to prepare a bacterial-cellsuspension.

Example 3

Improved Nitrile Hydratase Activity

The nitrile hydratase activity in the obtained bacterial-cell suspensionwas measured by the following method: 0.2 mL of the bacterial-cellmixture and 4.8 mL of a 50 mM phosphate buffer (pH 7.0) were mixed, towhich 5 mL of a 50 mM phosphate buffer (pH 7.0) containing 5.0% (w/v)acrylonitrile was further added. Next, the mixture was reacted whilebeing shaken at 10° C. for 10 minutes. Then, bacterial cells werefiltered and the amount of produced acrylamide was determined using gaschromatography.

<Analysis Conditions>

analysis instrument: gas chromatograph GC-14B (Shimadzu Corporation)detector: FID (detection at 200° C.) column: 1 m glass column filledwith PoraPak PS (column filler made by Waters Corp.) column temperature:190° C.

Nitrile hydratase activity was determined by conversion from the amountof acrylamide. Here, regarding nitrile hydratase activity, the amount ofenzyme to produce 1 μmol of acrylamide per 1 minute is set as 1 U. Table7 shows relative activities when the parent strain activity withoutamino-acid substitution was set at 1.0.

TABLE 7 Measurement results of catalytic activity amino-acid name ofcatalytic activity substitution plasmid (relative value) none (parentstrain) pSJ034 1.0 (comp. example) Wβ48D pSJ103 1.2 Wβ48E pSJ104 1.6Wβ48K pSJ109 1.1 Wβ48M pSJ111 3.1 Wβ48N pSJ112 1.8 Wβ48P pSJ113 2.0Wβ48S pSJ116 1.1 Wβ48T pSJ117 1.3

From the results above, enhanced enzymatic activity was confirmed in theimproved nitrile hydratase in which an amino acid at position 48 in theβ subunit was substituted with aspartic acid, lysine, asparagine,proline, serine or threonine.

Example 4

Preparation and Evaluation of Improved Nitrile Hydratase

Plasmid pFR005 formed in preparation example 2 as a template plasmid wasused to substitute an amino acid at position 48 of the β subunit.

Namely, using the method in example 1, each of the improved nitrilehydratases with a substituted amino acid were prepared, and atransformant was obtained by the method in example 2. Further, theenzymatic activity was measured by the same method in example 3. Theresults are shown in Table 8.

TABLE 8 Measurement results of catalytic activity name of amino-acidcatalytic activity plasmid substitution (relative value) pFR005 Pβ17G,Sβ57K, Tβ107K, Kβ114Y, 1.0 (comp. example) Nβ167S, Vβ219A pER1102 Pβ17G,Sβ57K, Tβ107K, Kβ114Y, 1.6 Nβ167S, Vβ219A, Wβ48C pER1103 Pβ17G, Sβ57K,Tβ107K, Kβ114Y, 1.7 Nβ167S, Vβ219A, Wβ48D pER1104 Pβ17G, Sβ57K, Tβ107K,Kβ114Y, 1.3 Nβ167S, Vβ219A, Wβ48E pER1107 Pβ17G, Sβ57K, Tβ107K, Kβ114Y,1.2 Nβ167S, Vβ219A, Wβ48H pER1108 Pβ17G, Sβ57K, Tβ107K, Kβ114Y, 1.6Nβ167S, Vβ219A,Wβ48I pER1109 Pβ17G, Sβ57K, Tβ107K, Kβ114Y, 1.4 Nβ167S,Vβ219A, Wβ48K pER1112 Pβ17G, Sβ57K, Tβ107K, Kβ114Y, 3.7 Nβ167S, Vβ219A,Wβ48M pER1113 Pβ17G, Sβ57K, Tβ107K, Kβ114Y, 1.7 Nβ167S, Vβ219A, Wβ48NpER1114 Pβ17G, Sβ57K, Tβ107K, Kβ114Y, 1.7 Nβ167S, Vβ219A, Wβ48P pER1116Pβ17G, Sβ57K, Tβ107K, Kβ114Y, 1.9 Nβ167S, Vβ219A, Wβ48Q pER1117 Pβ17G,Sβ57K, Tβ107K, Kβ114Y, 1.8 Nβ167S, Vβ219A, Wβ48S pER1119 Pβ17G, Sβ57K,Tβ107K, Kβ114Y, 1.1 Nβ167S, Vβ219A, Wβ48T

From the results above, the same enzymatic activity was confirmed in themutant nitrile hydratase when the amino acid at X₄ (corresponding to anamino acid at position 48 in the β subunit) in the amino-acid sequenceshown in SEQ ID NO: 50 was substituted with an amino acid selected fromamong cysteine, glutamic acid, aspartic acid, histidine, isoleucine,lysine, methionine, asparagine, proline, glutamine, serine andthreonine.

Example 5

SDS-Polyacrylamide Gel Electrophoresis

Using a sonicator VP-300 (TAITEC Corporation), the bacterial-cellsuspension prepared in example 2 was homogenized for 10 minutes whilebeing ice-cooled. Next, the bacterial-cell homogenate was centrifuged at13500 rpm for 30 minutes and a cell-free extract was obtained from thesupernatant. After the protein content of the cell extract was measuredusing a Bio-Rad protein assay kit, the cell extract was mixed with apolyacrylamide gel electrophoresis sample buffer (0.1 M Tris-HCl (pH6.8), 4% w/v SDS, 12% v/v β mercaptoethanol, 20% v/v glycerol, and atrace of bromophenol blue), and boiled for 5 minutes for denaturation. A10% acrylamide gel was prepared and denatured samples were applied tohave an equivalent protein mass per one lane to conduct electrophoresisanalysis (FIG. 4).

As a result, since hardly any difference was observed in the bandstrength of nitrile hydratase in all the samples, the expressed amountof nitrile hydratase was found to be the same. Accordingly, theenzymatic specific activity was found to be attributed to the improvedenzymatic activity.

Example 6

Preparation of Transformant Containing Nitrile Hydratase Derived fromRhodococcus Rhodocrous M8 Strain (Hereinafter Referred to as M8 Strain)

(1) Preparation of Chromosomal DNA from MS Strain

The M8 strain (SU 1731814) is obtained from the Russian Institute ofMicroorganism Biochemistry and Physiology (VKPM S-926). In 100 mL of anMYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3%Bacto malt extract, 0.2% K₂HPO₄, 0.2% KH₂PO₄, pH 7.0), the M8 strain wassubjected to shaking culture at 30° C. for 72 hours. The culture mixturewas centrifuged, and the collected bacterial cells were suspended in 4mL of a Saline-EDTA solution (0.1 M EDTA, 0.15 M NaCl, pH 8.0). Then, 8mg of lysozyme was added to the suspension, which was shaken at 37° C.for 1˜2 hours and was frozen at −20° C.

Next, 10 mL of Tris-SDS solution (1% SDS, 0.1M NaCl, 0.1 M Tris-HCl (pH9.0)) was added to the suspension while the suspension was gentlyshaken. Proteinase K (Merck KGaA) was further added (final concentrationof 0.1 mg) and shaken at 37° C. for 1 hour. Next, an equivalent volumeof TE saturated phenol was added, agitated (TE: 10 mM Tris-HCl, 1 mMEDTA (pH 8.0)) and then centrifuged. The supernatant was collected and adouble volume of ethanol was added and DNA strands were wrapped around aglass rod. Then, the phenol was removed through centrifugation bysuccessively adding 90%, 80%, and 70% ethanol.

Next, the DNA was dissolved in a 3 mL TE buffer, to which a RibonucleaseA solution (processed at 100° C. for 15 minutes) was added to have a 10μg/mL concentration and shaken at 37C for 30 minutes. Proteinase K(Merck KGaA) was further added and shaken at 37° C. for 30 minutes.After an equivalent volume of TE saturated phenol was added andcentrifuged, the mixture was separated into upper and lower layers.

An equivalent volume of TE saturated phenol was further added to theupper layer and centrifuged to separate into upper and lower layers.Such a process was repeated. Then, an equivalent volume of chloroform(containing 4% isoamyl alcohol) was added, centrifuged and the upperlayer was collected. Then, a double volume of ethanol was added to theupper layer and the DNA strands were collected by wrapping them around aglass rod. Accordingly, chromosomal DNA was obtained.

(2) Using PCR, Preparation of Improved Nitrile Hydratase fromChromosomal DNA Derived from M8 Strain

The nitrile hydratase derived from the M8 strain is described in anon-patent publication (Veiko, V. P. et al., “Cloning, NucleotideSequence of Nitrile Hydratase Gene from Rhodococcus rhodochrous M8,”Russian Biotechnology (Mosc.) 5, 3-5 (1995)). The sequences of βsubunit, α subunit and activator are respectively identified in SEQ IDNOs: 37, 38 and 39. Based on the sequence information, primers of SEQ IDnumbers 40 and 41 in the sequence listing were synthesized and PCR wasperformed using the chromosomal DNA prepared in step (1) above as atemplate.

<Composition of PCR Reaction Mixture>

sterile water 20 μL template DNA (chromosomal DNA) 1 μL primer M8-1 (10mM) 2 μL primer M8-2 (10 mM) 2 μL PrimeSTAR MAX (2×) 25 μL total 50 μL<Primers>

M8-1: (SEQ ID NO: 40) GGTCTAGAATGGATGGTATCCACGACACAGGC M8-2:(SEQ ID NO: 41) cccctgcaggtcagtcgatgatggccatcgattc<Reaction Conditions>(98° C. for 10 sec, 55° C. for 5 sec, 72° C. for 30 sec)×30 cycles

After the completion of PCR, 5 μL of the reaction mixture was subjectedto 0.7% agarose gel electrophoresis (0.7 wt. % Agarose I, made by DojinChemical Co., Ltd.) and an amplified fragment of 1.6 kb was detected.The reacted mixture was purified using Wizard SV gel and PCR Clean-UpSystem (Promega KK).

Next, the collected PCR product was coupled with a vector (pUC118/HincII site) using a ligation kit (made by Takara Shuzo Co., Ltd.) so thatcompetent cells of E. coli JM109 were transformed using the reactionmixture. A few clones from the obtained transformant colony wereinoculated into 1.5 mL of an LB-Amp culture medium, and incubated at 37°C. for 12 hours while being shaken. After incubation was finished, thebacterial cells were collected from the culture through centrifugation.Plasmid DNA was extracted from the collected bacterial cells usingQIAprep Spin Miniprep Kit (Qiagen). The base sequence of nitrilehydratase in the obtained plasmid DNA was confirmed using a sequencingkit and automated sequencer CEQ 8000 (Beckman Coulter, Inc.) (SEQ ID NO:62).

Next, the obtained plasmid DNA was cleaved with restriction enzymes XbaIand Sse8387I, and subjected to 0.7% agarose gel electrophoresis so as tocollect a nitrile hydratase gene fragment (1.6 kb), which was theninserted into XbaI-Sse8387I site of plasmid pSJ042. The obtained plasmidwas named pSJ-N01A. Here, pSJ042 as a plasmid capable of expressingnitrile hydratase in Rhodococcus J1 strain was prepared by a methoddescribed in JP publication 2008-154552 (the content is incorporated inthis application by reference). Plasmid pSJ023 used for preparation ofpSJ042 is registered as transformant ATCC 12674/pSJ023 (FERM BP-6232) atthe International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology (Central 6, 1-1-1 Higashi,Tsukuba, Ibaraki), deposited Mar. 4, 1997.

Example 7

Preparation and Evaluation of Improved Nitrile Hydratase

Using plasmid pSJ-N01A obtained in example 6, the amino acid at position48 of the β subunit was substituted. The same method in example 1 wasemployed for amino-acid substitution to prepare an improved nitrilehydratase. Next, using the same method in example 3, a transformant ofRhodococcus rhodocrous ATCC 12674 strain and its bacterial-cellsuspension were prepared. Then, the enzymatic activity was measured bythe same method as in example 4. The results are shown in Table 9.

TABLE 9 Measurement results of catalytic activity name of amino-acidcatalytic activity plasmid substitution (relative value) pSJ-N01A none(parent strain) 1.0 (comp. example) pSJR13 Wβ48M 2.4 pSJR21 Wβ48N 2.3

From the results in table 9, when the amino acid at position 48 of the03 subunit was substituted, the enzymatic activity of the improvednitrile hydratase was confirmed to be enhanced the same as in example 3.

Example 8

Preparation of Improved Nitrile Hydratase

Using plasmid pSJ034 formed in preparation example 1, amino-acidsubstitution was conducted. The following composition of reactionmixture, reaction conditions and primers shown in table 2 were used forthe PCR.

<Composition of PCR Reaction Mixture>

sterile water 20 μL pSJ034 (1 ng/mL) 1 μL Forward primer (10 mM) 2 μLReverse primer (10 mM) 2 μL PrimeSTAR MAX (2×) 25 μL total 50 μL<PCR Reaction Conditions>(98° C. for 10 sec, 55° C. for 5 sec, 72° C. for 90 sec)×30 cycles<Primers>

TABLE 10 substi- tuted SEQ amino name of ID acid primer sequence NO Aβ37A-F GTCAATTGCGACTTGGATGCATCTCAAG 67 β37A-RCCAAGTCGCAATTGACAGGGTCCGACC 68 D β37D-F GTCAATTGACACTTGGATGCATCTCAAG 69β37D-R CCAAGTGTCAATTGACAGGGTCCGACC 70 F β37F-FGTCAATTTTCACTTGGATGCATCTCAAG 71 β37F-R CCAAGTGAAAATTGACAGGGTCCGACC 72 Iβ37I-F GTCAATTATCACTTGGATGCATCTCAAG 73 β37I-RCCAAGTGATAATTGACAGGGTCCGACC 74 M β37M-F GTCAATTATGACTTGGATGCATCTCAAG 75β37M-R CCAAGTCATAATTGACAGGGTCCGACC 76 T β37T-FGTCAATTACCACTTGGATGCATCTCAAG 77 β37T-R CCAAGTGGTAATTGACAGGGTCCGACC 78 Vβ37V-F GTCAATTGTCACTTGGATGCATCTCAAG 79 β37V-RCCAAGTGACAATTGACAGGGTCCGACC 80

After the completion of PCR, 5 μL of the reaction mixture was subjectedto 0.7% agarose gel electrophoresis and an amplified fragment of 1 kbwas confirmed. Then, 1 L of DpnI (provided with a kit) was added to thePCR reaction mixture and reacted at 37° C. for an hour to remove thetemplate plasmid. The reacted mixture was purified using Wizard SV geland PCR Clean-Up System (Promega), and JM109 was transformed using thepurified PCR reaction product. Then, a plasmid DNA was extracted fromthe obtained culture using QIAprep Spin Miniprep Kit (Qiagen), and thebase sequence of the nitrile hydratase was confirmed using an automatedsequencer CEQ 8000 (Beckman Coulter, Inc.) Obtained plasmids were namedas shown in Table 11.

TABLE 11 name of amino-acid plasmid substitution pSJ120 Lβ37A pSJ122Lβ37D pSJ124 Lβ37F pSJ127 Lβ37I pSJ129 Lβ37L pSJ130 Lβ37M pSJ136 Lβ37TpSJ137 Lβ37V

Example 9

Preparation of Rhodococcus Transformant

Cells of Rhodococcus rhodocrous ATCC 12674 strain in a logarithmicgrowth phase were collected using a centrifuge, washed three times withice-cold sterile water, and suspended in the sterile water. Then, 1 μLof plasmid prepared in example 1 and 10 μL of the bacterial-cellsuspension were mixed and ice-cooled. The DNA and the bacterial-cellsuspension were supplied in a cuvette, and electric pulse treatment wasconducted using an electroporation device, Gene Pulser (Bio-RadLaboratories), under conditions of 2.0 kV and 200Ω. The electric-pulseprocessed mixture was let stand in an ice-cold condition for 10 minutes,and subjected to heat shock at 37° C. for 10 minutes. After 500 μL of anMYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3%Bacto malt extract, 0.2% K₂HPO₄, 0.2% KH₂PO₄) was added and let stand at30° C. for 5 hours, and applied onto an MYK agar culture mediumcontaining 50 μg/mL kanamycin and incubated at 30° C. for 3 days. Theobtained colony after incubating at 30° C. for 3 days was used as atransformant.

Each transformant obtained above was inoculated into an MYK culturemedium (50 g/mL kanamycin), and subjected to shaking culture at 30° C.for 2 days. Then, 1% culture was each inoculated into a GGPK culturemedium (1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05%K₂HPO₄, 0.05% KH₂PO₄, 0.05% Mg₂O₄.7H₂O, 1% CoCl₂, 0.1% urea, 50 μg/mLkanamycin, pH 7.2), and shaking culture was performed at 30° C. for 3days. Bacterial cells were collected by using a centrifuge and werewashed with a 100 mM phosphate buffer (pH 7.0) to prepare abacterial-cell suspension.

Example 10

Improved Nitrile Hydratase Activity

The nitrile hydratase activity in the obtained bacterial-cell suspensionwas measured by the following method: 0.2 mL of the bacterial-cellmixture and 4.8 mL of a 50 mM phosphate buffer (pH 7.0) were mixed, towhich 5 mL of a 50 mM phosphate buffer (pH 7.0) containing 5.0% (w/v)acrylonitrile was further added. Next, the mixture was reacted whilebeing shaken at 10° C. for 10 minutes. Then, bacterial cells werefiltered and the amount of produced acrylamide was determined using gaschromatography.

<Analysis Conditions>

-   analysis instrument: gas chromatograph GC-14D (Shimadzu Corporation)-   detector: FID (detection at 200° C.)-   column: 1 m glass column filled with PoraPak PS (column filler made    by Waters Corp.)-   column temperature: 190° C.

Nitrile hydratase activity was determined by conversion from the amountof acrylamide. Here, regarding nitrile hydratase activity, the amount ofenzyme to produce 1 μmol of acrylamide per 1 minute is set as 1 U. Table12 shows relative activities when the parent strain activity withoutamino-acid substitution was set at 1.0.

TABLE 12 amino-acid name of catalytic activity substitution plasmid(relative value) none (parent strain) pSJ034 1.0 (comp. example) Lβ37ApSJ120 1.3 Lβ37D pSJ122 1.5 Lβ37F pSJ124 1.2 Lβ37I pSJ127 1.2 Lβ37MpSJ130 1.2 Lβ37T pSJ136 1.2 Lβ37V pSJ137 1.3

From the results above, enhanced enzymatic activity was confirmed in theenzyme in which an amino acid at position 37 in the β subunit wassubstituted with an amino acid selected from among alanine, valine,asparagine, threonine, phenylalanine, isoleucine and methionine.

Example 11

SDS-Polyacrylamide Gel Electrophoresis

Using a sonicator VP-300 (TAITEC Corporation), the bacterial-cellsuspension prepared in example 2 was homogenized for 10 minutes while itwas ice-cooled. Next, the bacterial-cell homogenate was centrifuged at13500 rpm for 30 minutes and a cell-free extract was obtained from thesupernatant. After the protein content of the cell extract was measuredusing a Bio-Rad protein assay kit, the cell extract was mixed with apolyacrylamide gel electrophoresis sample buffer (0.1 M Tris-HCl (pH6.8), 4% w/v of SDS, 12% v/v of 0 mercaptoethanol, 20% v/v of glycerol,and a trace of bromophenol blue), and boiled for 5 minutes fordenaturation. A 10% acrylamide gel was prepared, and denatured sampleswere applied to have an equivalent protein mass per one lane to conductelectrophoresis analysis.

As a result, since hardly any difference was observed in the bandstrength of nitrile hydratase in all the samples, the expressed amountof nitrile hydratase was found the same. Accordingly, enzymatic specificactivity was found to be attributed to be the improved enzymaticactivity.

Example 12

Preparation and Evaluation of Improved Nitrile Hydratase

Plasmid pFR005 below was used as a template plasmid substitute an aminoacid at position 37 of the β subunit.

Namely, using the method in example 1, an improved nitrile hydratasewith a substituted amino acid was prepared, and a transformant ofRhodococcus rhodocrous ATCC 12674 strain and its bacterial-cellsuspension were obtained by the method in example 2. Further, theenzymatic activity was measured by the same method in example 3. Theresults are shown in Table 13.

TABLE 13 catalytic activity name of plasmid amino-acid substitution(relative value) pFR005 Pβ17G, Sβ57K, Nβ167S, Tβ107K, 1.0 (comp. Kβ114Y,Vβ219A example) pER1121 Pβ17G, Sβ57K, Nβ167S, Tβ107K, 1.6 Kβ114Y,Vβ219A, Lβ37A pER1140 Pβ17G, Sβ57K, Nβ167S, Tβ107K, 1.3 Kβ114Y, Vβ219A,Lβ37D

From the results above, the amino-acid substitution according to thepresent invention applies not only to a wild-type nitrile hydratase butto a mutant nitrile hydratase to exhibit the same effects.

Example 13

Preparation of Improved Nitrile Hydratase

Using pSJ034 formed in preparation example 1, amino-acid substitutionwas conducted. The following composition of a reaction mixture, reactionconditions and primers shown in Table 14 were used for the PCR.

<Composition of PCR Reaction Mixture>

sterile water 20 μL pSJ034 (1 ng/mL)  1 μL Forward primer (10 mM)  2 μLReverse primer (10 mM)  2 μL PrimeSTAR MAX (2×) 25 μL total 50 μL<PCR Reaction Conditions>(98° C. for 10 sec, 55° C. for 5 sec, 72° C. for 90 sec)×30 cycles<Primers>

TABLE 14 substi- tuted SEQ amino name of ID acid primer Sequence NO Aα83A-F GGTGAGGCGGCACACCAAATTTCGGCG  83 α83A-R GTGTGCCGCCTCACCGGCATAGCCC 84 C α83C-F GGTGAGTGCGCACACCAAATTTCGGCG  85 α83C-RGTGTGCGCACTCACCGGCATAGCCC  86 D α83D-F GGTGAGGACGCACACCAAATTTCGGCG  87α83D-R GTGTGCGTCCTCACCGGCATAGCCC  88 E α83E-FGGTGAGGAGGCACACCAAATTTCGGCG  89 α83E-R GTGTGCCTCCTCACCGGCATAGCCC  90 Fα83F-F GGTGAGTTCGCACACCAAATTTCGGCG  91 α83F-R GTGTGCGAACTCACCGGCATAGCCC 92 G α83G-F GGTGAGGGCGCACACCAAATTTCGGCG  93 α83G-RGTGTGCGCCCTCACCGGCATAGCCC  94 H α83H-F GGTGAGCACGCACACCAAATTTCGGCG  95α83H-R GTGTGCGTGCTCACCGGCATAGCCC  96 M α83M-FGGTGAGATGGCACACCAAATTTCGGCG  97 α83M-R GTGTGCCATCTCACCGGCATAGCCC  98 Pα83P-F GGTGAGCCGGCACACCAAATTTCGGCG  99 α83P-R GTGTGCCGGCTCACCGGCATAGCCC100 S α83S-F GGTGAGTCCGCACACCAAATTTCGGCG 101 α83S-RGTGTGCGGACTCACCGGCATAGCCC 102 T α83T-F GGTGAGACCGCACACCAAATTTCGGCG 103α83T-R GTGTGCGGTCTCACCGGCATAGCCC 104

After the completion of PCR, 5 μL of the reaction mixture was subjectedto 0.7% agarose gel electrophoresis and an amplified fragment of 11 kbwas confirmed. Then, 1 μL of DpnI (provided with a kit) was added to thePCR reaction mixture and reacted at 37C for an hour to remove thetemplate plasmid. The reacted mixture was purified using Wizard SV geland PCR Clean-Up System (Promega), and JM109 was transformed using thepurified PCR reaction product. Then, a plasmid DNA was extracted fromthe obtained culture using QIAprep Spin Miniprep Kit (Qiagen), and thebase sequence of the nitrile hydratase was confirmed using an automatedsequencer CEQ 8000 (Beckman Coulter, Inc.) Obtained plasmids were namedas shown in Table 15.

TABLE 15 name of plasmid amino-acid substitution pSJ127 Qα83A pSJ152Qα83C pSJ153 Qα83D pSJ154 Qα83E pSJ155 Qα83F pSJ156 Qα83G pSJ157 Qα83HpSJ130 Qα83M pSJ132 Qα83N pSJ159 Qα83P pSJ161 Qα83S pSJ162 Qα83T

Example 14

Preparation of Rhodococcus Transformant

Cells of Rhodococcus rhodocrous strain ATCC 12674 in a logarithmicgrowth phase were collected using a centrifuge, washed three times withice-cold sterile water, and suspended in the sterile water. Then, 1 μLof plasmid prepared in example 1 and 10 μL of the bacterial-cellsuspension were mixed and ice-cooled. The DNA and the bacterial-cellsuspension were supplied in a cuvette, and electric pulse treatment wasconducted using an electroporation device, Gene Pulser (Bio-RadLaboratories), under conditions of 2.0 kV and 200Ω. The electric-pulseprocessed mixture was let stand in an ice-cold condition for 10 minutes,and subjected to heat shock at 37° C. for 10 minutes. After 500 μL of anMYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3%Bacto malt extract, 0.2% K₂HPO₄, 0.2% KH₂PO₄) was added and let stand at30° C. for 5 hours, and applied onto an MYK agar culture mediumcontaining 50 μg/mL kanamycin and incubated at 30° C. for 3 days. Theobtained colony after incubating at 30° C. for 3 days was used as atransformant.

Each transformant obtained above were inoculated into an MYK culturemedium (50 μg/mL kanamycin), and subjected to shaking culture at 30° C.for 2 days. Then, 1% culture was inoculated into a GGPK culture medium(1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K₂HPO₄,0.05% KH₂P O₄, 0.05% Mg₂O₄.7H₂O, 1% CoCl₂, 0.1% urea, 50 μg/mLkanamycin, pH 7.2), and shaking culture was performed at 30° C. for 3days. Then, bacterial cells were collected by using a centrifuge andwere washed with a 100 mM phosphate buffer (pH 7.0) to prepare abacterial-cell suspension.

Example 15

Improved Nitrile Hydratase Activity

The nitrile hydratase activity in the obtained bacterial-cell suspensionwas measured by the following method: 0.2 mL of the bacterial-cellmixture and 4.8 mL of a 50 mM phosphate buffer (pH 7.0) were mixed, towhich 5 mL of a 50 mM phosphate buffer (pH 7.0) containing 5.0% (w/v)acrylonitrile was further added. Next, the mixture was reacted whilebeing shaken at 10° C. for 10 minutes. Then, bacterial cells werefiltered and the amount of produced acrylamide was determined using gaschromatography.

<Analysis Conditions>

-   analysis instrument: gas chromatograph GC-14B (Shimadzu Corporation)-   detector: FID (detection at 200° C.)-   column: 1 m glass column filled with PoraPak PS (column filler made    by Waters Corp.)-   column temperature: 190° C.

Nitrile hydratase activity was determined by conversion from the amountof acrylamide. Here, regarding nitrile hydratase activity, the amount ofenzyme to produce 1 μmol of acrylamide per 1 minute is set as 1 U. Table16 shows relative activities when the parent strain activity withoutamino-acid substitution was set at 1.0.

TABLE 16 catalytic activity name of plasmid amino-acid substitution(relative value) pSJ034 none (parent strain) 1.0 (comp. example) pSJ127Qα83A 5.3 pSJ152 Qα83C 3.7 pSJ153 Qα83D 1.9 pSJ154 Qα83E 1.2 pSJ155Qα83F 1.8 pSJ156 Qα83G 4.4 pSJ157 Qα83H 1.9 pSJ130 Qα83M 2.3 pSJ132Qα83N 5.7 pSJ159 Qα83P 1.5 pSJ161 Qα83S 5.8 pSJ162 Qα83T 3.8

From the results above, enhanced enzymatic activity was confirmed in theenzyme in which an amino acid at position 83 in the α subunit wassubstituted with an amino acid selected from among alanine, asparticacid, phenylalanine, histidine, methionine and asparagine.

Example 16

Preparation and Evaluation of Improved Nitrile Hydratase

Plasmid pFR005 formed below was used as a template plasmid to substitutean amino acid at position 83 of the α subunit.

Namely, using the method in example 1, an improved nitrile hydratasewith a substituted amino acid was prepared, and a transformant ofRhodococcus rhodocrous strain ATCC 12674 and its bacterial-cellsuspension were obtained by the method in example 2. Further, theenzymatic activity was measured by the same method in example 3. Theresults are shown in Table 17.

TABLE 17 name of catalytic activity plasmid amino-acid substitution(relative value) pFR005 Pβ17G, Sβ57K, Tβ107K, Kβ114Y, 1.0 (comp.example) Nβ167S, Vβ219A pER1127 Pβ17G, Sβ57K, Tβ107K, Kβ114Y, 5.1Nβ167S, Vβ219A, Qα83A pER1129 Pβ17G, Sβ57K, Tβ107K, Kβ114Y, 1.9 Nβ167S,Vβ219A, Qα37L pER1130 Pβ17G, Sβ57K, Tβ107K, Kβ114Y, 2.7 Nβ167S, Vβ219A,Qα83M pER1132 Pβ17G, Sβ57K, Tβ107K, Kβ114Y, 4.8 Nβ167S, Vβ219A, Qα37N

From the results above, the amino-acid substitution according to thepresent invention applies not only to a wild-type nitrite hydratase butto a mutant nitrile hydratase to exhibit the same effects.

Example 17

Preparation of Transformant Containing Nitrile Hydratase Derived fromRhodococcus Rhodocrous M8 Strain (Hereinafter Referred to as M8 Strain)

(1) Preparation of Chromosomal DNA from M8 Strain

The M8 strain (SU 1731814) is obtained from Russian Institute ofMicroorganism Biochemistry and Physiology (VKPM S-926). In a 100 mL MYKculture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bactomalt extract, 0.2% K₂HPO₄, 0.2% KH₂PO₄, pH 7.0), the M8 strain wassubjected to shaking culture at 30° C. for 72 hours. The culture mixturewas centrifuged, and the collected bacterial cells were suspended in 4mL of Saline-EDTA solution (0.1 M EDTA, 0.15 M NaCl, pH 8.0). Then, 8 mgof lysozyme was added to the suspension, which was shaken at 37° C. for1˜2 hours and was frozen at −20° C.

Next, 10 mL of Tris-SDS solution (1% SDS, 0.1M NaCl, 0.1 M Tris-HCl (pH9.0)) was added to the suspension while the suspension was gentlyshaken. Proteinase K (Merck KGaA) was further added (final concentrationof 0.1 mg) and shaken at 37° C. for 1 hour. Next, an equivalent volumeof TE saturated phenol was added, agitated (TE: 10 mM Tris-HCl, 1 mMEDTA (pH 8.0)) and then centrifuged. The supernatant was collected, adouble volume of ethanol was added and DNA strands were wrapped around aglass rod. Then, the phenol was removed through centrifugation bysuccessively adding 90%, 80%, and 70% ethanol.

Next, the DNA was dissolved in a 3 mL TE buffer, to which a RibonucleaseA solution (processed at 100° C. for 15 minutes) was added to have a 10μg/mL concentration and shaken at 37° C. for 30 minutes. Proteinase K(Merck KGaA) was further added and shaken at 37° C. for 30 minutes.After an equivalent volume of TE saturated phenol was added andcentrifuged, the mixture was separated into upper and lower layers.

An equivalent volume of TE saturated phenol was further added to theupper layer and centrifuged to separate into upper and lower layers.Such a process was repeated. Then, an equivalent volume of chloroform(containing 4% isoamyl alcohol) was added, centrifuged and the upperlayer was collected. Then, a double volume of ethanol was added and theDNA strands were collected by wrapping them around a glass rod.Accordingly, chromosomal DNA was obtained.

(2) Using PCR, Preparation of Improved Nitrile Hydratase fromChromosomal DNA Derived from the M8 Strain

The nitrile hydratase derived from the M8 strain is described in anon-patent publication (Veiko, V. P. et al., “Cloning, NucleotideSequence of Nitrile Hydratase Gene from Rhodococcus rhodochrous M8,”Russian Biotechnology (Mosc.) 5, 3-5 (1995)). The sequences of 03subunit and α subunit are respectively identified as SEQ ID NOs: 17 and18. Based on the sequence information, primers of SEQ ID NOs: 115 and116 in the sequence listing were synthesized and PCR was performed usingthe chromosomal DNA prepared in step (1) above as a template.

<Composition of PCR Reaction Mixture>

sterile water 20 μL template DNA (chromosomal DNA)  1 μL primer M8-1 (10mM)  2 μL primer M8-2 (10 mM)  2 μL PrimeSTAR MAX (2×) 25 μL total 50 μL<Primers>

M8-1: (SEQ ID NO: 115) GGTCTAGAATGGATGGTATCCACGACACAGGC M8-2:(SEQ ID NO: 116) CCCCTGCAGGTCAGTCGATGATGGCCATCGATTC<PCR Reaction Conditions>(98° C. for 10 sec, 55° C. for 5 sec, 72° C. for 30 sec)×30 cycles

After completion of PCR, 5 μL of the reaction mixture was subjected to0.7% agarose gel electrophoresis (0.7 wt. % Agarose I, made by DojinChemical Co., Ltd.) and an amplified fragment of 1.6 kb was detected.The reacted mixture was purified using Wizard SV gel and PCR Clean-UpSystem (Promega KK).

Next, the collected PCR product was coupled with a vector (pUC118/HincII site) using a ligation kit (made by Takara Shuzo Co., Ltd.) so thatcompetent cells of E. coli JM109 were transformed using the reactionmixture. A few clones from the obtained transformant colonies wereinoculated into 1.5 mL of an LB-Amp culture medium, and subjected toshaking culture at 37° C. for 12 hours. After incubation was finished,the bacterial cells were collected from the culture throughcentrifugation. A plasmid DNA was extracted from the collected bacterialcells using QIAprep Spin Miniprep Kit (Qiagen). The base sequence ofnitrile hydratase in the obtained plasmid DNA was confirmed using asequencing kit and automated sequencer CEQ 8000 (Beckman Coulter, Inc.).

Next, the obtained plasmid DNA was cleaved at restriction enzyme XbaIand Sse8387I, and subjected to 0.7% agarose gel electrophoresis so as tocollect nitrile hydratase gene fragments (1.6 kb), which were thenintroduced into XbaI-Sse8387I site of plasmid pSJ042. The obtainedplasmid was named pSJ-N01A. Here, pSJ042 as a plasmid capable ofexpressing nitrile hydratase in Rhodococcus J1 strain was prepared by amethod described in JP publication 2008-154552. Plasmid pSJ023 used forpreparation of pSJ042 is registered as transformant ATCC 12674/pSJ023(FERM BP-6232) at the International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (Central 6,1-1-1 Higashi, Tsukuba, Ibaraki), deposited Mar. 4, 1997.

Example 18

Preparation and Evaluation of Improved Nitrile Hydratase

Using plasmid pSJ-N01A obtained in example 5, the amino acid at position83 of the α subunit was substituted. The same method as in example 1 wasemployed for amino-acid substitution to prepare an improved nitrilehydratase. Next, using the same method as in example 2, a transformantof Rhodococcus rhodocrous ATCC 12674 strain and its bacterial-cellsuspension were prepared. Then, the enzymatic activity was measured bythe same method as in example 4. The results are shown in Table 18.

TABLE 18 catalytic activity name of plasmid amino-acid substitution(relative value) pSJ-N01A none (parent strain) 1.0 (comp. example)pSJR17 Qα83M 6.9

From the results above, pSJR17 in which the amino acid at position 83 ofthe a subunit was substituted with methionine was found to have anenhanced enzymatic activity the same as in example 4.

Example 19

Preparation of Improved Nitrile Hydratase

Using plasmid pSJ034 formed in preparation example 1, amino-acidsubstitution was conducted. The following composition of a reactionmixture, reaction conditions and primers were used for the PCR.

<Composition of PCR Reaction Mixture>

sterile water 20 μL pSJ034 (1 ng/mL)  1 μL Forward primer (10 mM)  2 μLReverse primer (10 mM)  2 μL PrimeSTAR MAX (2×) 25 μL total 50 μL<PCR Reaction Conditions>(98° C. for 10 sec, 55° C. for 5 sec, 72° C. for 90 sec)×30 cycles<Primers> Saturation Mutagenesis for α82

α82RM-F: (SEQ ID NO: 129) ATGCCGGTNNSCAGGCACACCAAATTT α82RM-R:(SEQ ID NO: 130) TGTGCCTGSNNACCGGCATAGCCCAAT

After the completion of PCR, 5 μL of the reaction mixture was subjectedto 0.7% agarose gel electrophoresis and an amplified fragment of 1 kbwas confirmed. Then, 1 μL of DpnI (provided with a kit) was added to thePCR reaction mixture and reacted at 37° C. for an hour to remove thetemplate plasmid. The reacted mixture was purified using Wizard SV geland PCR Clean-Up System (Promega), and JM109 was transformed using thepurified PCR reaction product. Then, a plasmid DNA was extracted fromthe obtained culture using QIAprep Spin Miniprep Kit (Qiagen), and thebase sequence of the nitrile hydratase was confirmed using an automatedsequencer CEQ 8000 (Beckman Coulter, Inc.) Obtained plasmids were namedas shown in Table 19.

TABLE 19 name of plasmid amino-acid substitution pSJ173 Eα82C pSJ174Eα82F pSJ175 Eα82H pSJ176 Eα82I pSJ177 Eα82K pSJ178 Eα82M pSJ179 Eα82QpSJ180 Eα82R pSJ181 Eα82T pSJ182 Eα82Y

Example 20

Preparation of Rhodococcus Transformant

Cells of Rhodococcus rhodocrous ATCC 12674 strain in a logarithmicgrowth phase were collected using a centrifuge, washed three times withice-cold sterile water, and suspended in the sterile water. Then, 1 μLof plasmid prepared in example 2 and 10 μL of the bacterial-cellsuspension were mixed and ice-cooled. The DNA and the bacterial-cellsuspension were supplied in a cuvette, and electric pulse treatment wasconducted using an electroporation device, Gene Pulser (Bio-RadLaboratories), under conditions of 2.0 kV and 200Ω. The electric-pulseprocessed mixture was let stand in an ice-cold condition for 10 minutes,and subjected to heat shock at 37° C. for 10 minutes. After 500 μL of anMYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3%Bacto malt extract, 0.2% K₂HPO₄, 0.2% KH₂PO₄) was added and let stand at30° C. for 5 hours, and applied onto an MYK agar culture mediumcontaining 50 μg/mL kanamycin and incubated at 30° C. for 3 days. Theobtained colony after incubating at 30° C. for 3 days was used as atransformant.

Each transformant obtained above was inoculated into an MYK culturemedium (50 μg/mL kanamycin), subjected to shaking culture at 30° C. for2 days. Then, 1% culture was inoculated into a GGPK culture medium (1.5%glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K₂HPO₄, 0.05%KH₂PO₄, 0.05% Mg₂O₄.7H₂O, 1% CoCl₂, 0.1% urea, 50 μg/mL kanamycin, pH7.2), and subjected to shaking culture at 30° C. for 3 days. Then,bacterial cells were collected by using a centrifuge and were washedwith a 100 mM phosphate buffer (pH 7.0) to prepare a bacterial-cellsuspension.

Example 21

Improved Nitrile Hydratase Activity

The nitrile hydratase activity in the obtained bacterial-cell suspensionwas measured by the following method.

After 0.2 mL of the bacterial-cell mixture and 4.8 mL of a 50 mMphosphate buffer (pH 7.0) were mixed, 5 mL of a 50 mM phosphate buffer(pH 7.0) containing 5.0% (w/v) acrylonitrile was further added, and themixture was reacted while being shaken at 10° C. for 10 minutes. Then,bacterial cells were filtered and the amount of produced acrylamide wasdetermined by gas chromatography.

<Analysis Conditions>

-   analysis instrument: gas chromatograph GC2014 (Shimadzu Corporation)-   detector: FID (detection at 200° C.)-   column: 1 m glass column filled with PoraPak PS (column filler made    by Waters Corp.)-   column temperature: 190° C.

Nitrile hydratase activity was determined by conversion from the amountof acrylamide. Here, regarding nitrile hydratase activity, the amount ofenzyme to produce 1 μmol of acrylamide per 1 minute is set as 1 U. Table20 shows relative activities when the parent strain activity withoutamino-acid substitution was set at 1.0.

TABLE 20 catalytic activity name of plasmid amino-acid substitution(relative value) pSJ042 none (parent strain) 1.0 (comp. example) pSJ173Eα82C 2.6 pSJ174 Eα82F 4.3 pSJ175 Eα82H 1.3 pSJ176 Eα82I 3.6 pSJ177Eα82K 4.2 pSJ178 Eα82M 3.6 pSJ179 Eα82Q 2.3 pSJ180 Eα82R 4.2 pSJ181Eα82T 1.2 pSJ182 Eα82Y 2.1

From the results above, enhanced enzymatic activity was confirmed in theenzyme in which an amino acid at position 82 in the α subunit wassubstituted with an amino acid selected from among cysteine,phenylalanine, histidine, isoleucine, lysine, methionine, glutamine,arginine, threonine and tyrosine.

Example 22

SDS-Polyacrylamide Gel Electrophoresis

Using a sonicator VP-300 (TAITEC Corporation), the bacterial-cellsuspension prepared in example 3 was homogenized for 10 minutes whilebeing ice-cooled. Next, the bacterial-cell homogenate was centrifuged at13500 rpm for 30 minutes and a cell-free extract was obtained from thesupernatant. After the protein content of the cell extract was measuredusing a Bio-Rad protein assay kit, the cell extract was mixed with apolyacrylamide gel electrophoresis sample buffer (0.1 M Tris-HCl (pH6.8), 4% w/v SDS, 12% v/v 3 mercaptoethanol, 20% v/v glycerol, and atrace of bromophenol blue), and boiled for 5 minutes for denaturation. A10% acrylamide gel was prepared, and denatured samples were applied tohave an equivalent protein mass per one lane to conduct electrophoresisanalysis.

As a result, since hardly any difference was observed in the bandstrength of nitrile hydratase in all the samples, the expressed amountof nitrile hydratase was found the same. Accordingly, enzymatic specificactivity was found to be attributed to the improved enzymatic activity.

Example 23

Preparation of Improved Nitrile Hydratase

Using plasmid pSJ034 formed in preparation example 1, amino-acidsubstitution was conducted. The following composition of a reactionmixture, reaction conditions and primers were used for the PCR.

<Composition of PCR Reaction Mixture>

sterile water 20 μL pSJ034 (1 ng/mL)  1 μL Forward primer (10 mM)  2 μLReverse primer (10 mM)  2 μL PrimeSTAR MAX (2×) 25 μL total 50 μL<PCR Reaction Conditions>(98° C. for 10 sec, 55° C. for 5 sec, 72° C. for 90 sec)×30 cycles<Primers>Saturation Mutagenesis Primer for α85

α85RM-F: (SEQ ID NO: 133) CAGGCANNSCAAATTTCGGCGGTCTTC α85RM-R:(SEQ ID NO: 134) AATTTGSNNTGCCTGCTCACCGGCATA

After the completion of PCR, 5 μL of the reaction mixture was subjectedto 0.7% agarose gel electrophoresis and an amplified fragment of 1 kbwas confirmed. Then, 1 μL of DpnI (provided with a kit) was added to thePCR reaction mixture and reacted at 37° C. for an hour to remove thetemplate plasmid. The reacted mixture was purified using Wizard SV geland PCR Clean-Up System (Promega), and JM109 was transformed using thepurified PCR reaction product. Then, a plasmid DNA was extracted fromthe obtained culture using QIAprep Spin Miniprep Kit (Qiagen), and thebase sequence of the nitrile hydratase was confirmed using an automatedsequencer CEQ 8000 (Beckman Coulter, Inc.) Obtained plasmids were namedas shown in Table 21.

TABLE 21 name of plasmid amino-acid substitution pSJ165 Hα85C pSJ166Hα85E pSJ167 Hα85F pSJ168 Hα85I pSJ169 Hα85N pSJ170 Hα85Q pSJ171 Hα85SpSJ172 Hα85Y

Example 24

Preparation of Rhodococcus Transformant

Cells of Rhodococcus rhodocrous ATCC 12674 strain in a logarithmicgrowth phase were collected using a centrifuge, washed three times withice-cold sterile water, and suspended in the sterile water. Then, 1 μLof plasmid prepared in example 2 and 10 μL of the bacterial-cellsuspension were mixed and ice-cooled. The DNA and the bacterial-cellsuspension were supplied in a cuvette, and electric pulse treatment wasconducted using an electroporation device, Gene Pulser (Bio-RadLaboratories), under conditions of 2.0 kV and 200Ω. The electric-pulseprocessed mixture was let stand in an ice-cold condition for 10 minutes,and subjected to heat shock at 37° C. for 10 minutes. After 500 μL of anMYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3%Bacto malt extract, 0.2% K₂HPO₄, 0.2% KH₂PO₄) was added and let stand at30° C. for 5 hours, and applied onto an MYK agar culture mediumcontaining 50 μg/mL kanamycin and incubated at 30° C. for 3 days. Theobtained colony after incubating at 30° C. for 3 days was used as atransformant.

Each transformant obtained above was inoculated into an MYK culturemedium (50 μg/mL kanamycin), and subjected to shaking culture at 30° C.for 2 days. Then, 1% culture was each inoculated into a GGPK culturemedium (1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05%K₂HPO₄, 0.05% KH₂PO₄, 0.05% Mg₂O₄.7H₂O, 1% CoCl₂, 0.1% urea, 50 μg/mLkanamycin, pH 7.2), and shaking culture was performed at 30° C. for 3days. Bacterial cells were collected by using a centrifuge and werewashed with a 100 mM phosphate buffer (pH 7.0) to prepare abacterial-cell suspension.

Example 25

Improved Nitrile Hydratase Activity

The nitrile hydratase activity of the bacterial-cell suspension wasmeasure as follows.

After 0.2 mL of the bacterial-cell mixture and 4.8 mL of a 50 mMphosphate buffer (pH 7.0) were mixed, 5 mL of a 50 mM phosphate buffer(pH 7.0) containing 5.0% (w/v) acrylonitrile was further added, and themixture was reacted while being shaken at 10° C. for 10 minutes. Then,bacterial cells were filtered and the amount of produced acrylamide wasdetermined by gas chromatography.

<Analysis Conditions>

-   analysis instrument: gas chromatograph GC2014 (Shimadzu Corporation)-   detector: FID (detection at 200° C.)-   column: 1 m glass column filled with PoraPak PS (column filler made    by Waters Corp.)-   column temperature: 190° C.

Nitrile hydratase activity was determined by conversion from the amountof acrylamide. Here, regarding nitrile hydratase activity, the amount ofenzyme to produce 1 μmol of acrylamide per 1 minute is set as 1 U. Table22 shows relative activities when the parent strain activity withoutamino-acid substitution was set at 1.0.

TABLE 22 catalytic activity name of plasmid amino-acid substitution(relative value) pSJ042 none (parent strain) 1.0 (comp. example) pSJ165Hα85C 1.5 pSJ166 Hα85E 1.9 pSJ167 Hα85F 1.8 pSJ168 Hα85I 2.1 pSJ169Hα85N 2.3 pSJ170 Hα85Q 2.1 pSJ171 Hα85S 2.5 pSJ172 Hα85Y 1.5

From the results above, enhanced enzymatic activity was confirmed in theenzyme in which an amino acid at position 85 in the α subunit wassubstituted with an amino acid selected from among cysteine, glutamicacid, phenylalanine, isoleucine, asparagine, glutamine, serine andtyrosine.

Example 26

SDS-Polyacrylamide Gel Electrophoresis

Using a sonicator VP-300 (TAITEC Corporation), the bacterial-cellsuspension prepared in example 3 was homogenized for 10 minutes whilebeing ice-cooled. Next, the bacterial-cell homogenate was centrifuged at13500 rpm for 30 minutes and a cell-free extract was obtained from thesupernatant. After the protein content of the cell extract was measuredusing a Bio-Rad protein assay kit, the cell extract was mixed with apolyacrylamide gel electrophoresis sample buffer (0.1 M Tris-HCl (pH6.8), 4% w/v SDS, 12% v/v 3 mercaptoethanol, 20% v/v glycerol, and atrace of bromophenol blue), and boiled for 5 minutes for denaturation. A10% acrylamide gel was prepared, and denatured samples were applied tohave an equivalent protein mass per one lane to conduct electrophoresisanalysis.

As a result, since hardly any difference was observed in the bandstrength of nitrile hydratase in all the samples, the expressed amountof nitrile hydratase was found to be the same. Accordingly, theenzymatic specific activity was found to be attributed to the improvedenzymatic activity.

POTENTIAL INDUSTRIAL APPLICABILITY

According to the present invention, a novel improved (mutant) nitrilehydratase is provided with enhanced catalytic activity. Such an improvednitrile hydratase with enhanced catalytic activity is very useful toproduce amide compounds.

According to the present invention, a nitrile hydratase is obtained fromDNA encoding the improved nitrile hydratase above, a recombinant vectorcontaining the DNA, a transformant containing the recombinant vector,and a culture of the transformant, and a method for producing such anitrile hydratase is also provided. Moreover, a method for producing anamide compound using the protein (improved nitrile hydratase), theculture or the processed product of the culture is provided according tothe present invention.

According to the present invention, a novel improved (mutant) nitrilehydratase is provided with enhanced catalytic activity. Such an improvednitrile hydratase with enhanced catalytic activity is very useful toproduce amide compounds.

According to the present invention, a nitrile hydratase is obtained fromgenomic DNA encoding the improved nitrile hydratase above, a recombinantvector containing the genomic DNA, a transformant containing therecombinant vector, and a culture of the transformant, and a method forproducing such a nitrile hydratase is also provided. Moreover, a methodfor producing an amide compound using the protein (improved nitrilehydratase), the culture or the processed product of the culture isprovided according to the present invention.

ACCESSION NUMBERS

Rhodococcus rhodochrous J1 strain is internationally registered underaccession number “FERM BP-1478” at the International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology, (Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki), deposited Sep.18, 1987.

In addition, pSJ023 is a transformant “R. rhodochrous ATCC12674/pSJ023,” and is internationally registered under accession numberFERM BP-6232 at the International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science (Central 6, 1-1-1 Higashi,Tsukuba, Ibaraki), deposited Mar. 4, 1997.

[Description of Sequence Listing] SEQ ID NO: 1 base sequence of βsubunit derived from Rhodococcus rhodocrous J1 strain (FERM BP-1478) SEQID NO: 2 amino-acid sequence of β subunit derived from Rhodococcusrhodocrous J1 strain (FERM BP-1478) SEQ ID NO: 3 base sequence of αsubunit derived from Rhodococcus rhodocrous J1 strain (FERM BP-1478) SEQID NO: 4 amino-acid sequence of α subunit derived from Rhodococcusrhodocrous J1 strain (FERM BP-1478) SEQ ID NO: 5 amino-acid sequence ofβ subunit in Rhodococcus rhodocrous M8 (SU 1731814) SEQ ID NO: 6amino-acid sequence of β subunit in Rhodococcus ruber TH SEQ ID NO: 7amino-acid sequence of β subunit in Rhodococcus pyridinivorans MW33 (VKMAc-1515D) SEQ ID NO: 8 amino-acid sequence of β subunit in Rhodococcuspyridinivorans S85-2 SEQ ID NO: 9 amino-acid sequence of β subunit inRhodococcus pyridinivorans MS-38 SEQ ID NO: 10 amino-acid sequence of βsubunit in Nocardia sp.JBRs SEQ ID NO: 11 amino-acid sequence of βsubunit in Nocardia sp.YS-2002 SEQ ID NO: 12 amino-acid sequence of βsubunit in Rhodococcus rhodocrous ATCC 39384 SEQ ID NO: 13 β48C-F primerSEQ ID NO: 14 β48C-R primer SEQ ID NO: 15 β48D-F primer SEQ ID NO: 16β48D-R primer SEQ ID NO: 17 β48E-F primer SEQ ID NO: 18 β48E-R primerSEQ ID NO: 19 β48H-F primer SEQ ID NO: 20 β48H-R primer SEQ ID NO: 21β48I-F primer SEQ ID NO: 22 β48I-R primer SEQ ID NO: 23 β48K-F primerSEQ ID NO: 24 β48K-R primer SEQ ID NO: 25 β48M-F primer SEQ ID NO: 26β48M-R primer SEQ ID NO: 27 β48N-F primer SEQ ID NO: 28 β48N-R primerSEQ ID NO: 29 β48P-F primer SEQ ID NO: 30 β48P-R primer SEQ ID NO: 31β48Q-F primer SEQ ID NO: 32 β48Q-R primer SEQ ID NO: 33 β48S-F primerSEQ ID NO: 34 β48S-R primer SEQ ID NO: 35 β48T-F primer SEQ ID NO: 36β48T-R primer SEQ ID NO: 37 amino-acid sequence of β subunit in nitrilehydratase derived from M8 strain SEQ ID NO: 38 amino-acid sequence of αsubunit in nitrile hydratase derived from M8 strain SEQ ID NO: 39amino-acid sequence of activator in nitrile hydratase derived from M8strain SEQ ID NO: 40 M8-1 primer SEQ ID NO: 41 M8-2 primer SEQ ID NO: 42amino-acid sequence of β subunit in uncultured bacterium SP1 SEQ ID NO:43 amino-acid sequence of β subunit in uncultured bacterium BD2 SEQ IDNO: 44 amino-acid sequence of β subunit in Comamonas testosterone SEQ IDNO: 45 amino-acid sequence of β subunit in Geobacillusthermoglucosidasius Q6 SEQ ID NO: 46 amino-acid sequence of β subunit inPseudonocardia thermophila JCM 3095 SEQ ID NO: 47 amino-acid sequence ofβ subunit in Rhodococcus rhodocrous Cr4 SEQ ID NO: 48 amino-acidsequence of cysteine cluster of α subunit in iron-containing nitrilehydratase SEQ ID NO: 49 amino-acid sequence in cysteine cluster of αsubunit in cobalt-containing nitrile hydratase SEQ ID NO: 50predetermined amino-acid sequence to be used in the present inventionSEQ ID NO: 51 amino-acid sequence of β subunit related to the presentinvention SEQ ID NO: 52 amino-acid sequence of β subunit in Rhodococcusruber RH (CN 101463358) SEQ ID NO: 53 base sequence of nitrile hydrataseJ1D SEQ ID NO: 54 base sequence of nitrile hydratase 203 SEQ ID NO: 55base sequence of nitrile hydratase 414 SEQ ID NO: 56 base sequence ofnitrile hydratase 855 SEQ ID NO: 57 base sequence of the α subunit innitrile hydratase D2 SEQ ID NO: 58 base sequence of nitrile hydratase005 SEQ ID NO: 59 base sequence of nitrile hydratase 108A SEQ ID NO: 60base sequence of nitrile hydratase 211 SEQ ID NO: 61 base sequence ofnitrile hydratase 306A SEQ ID NO: 62 base sequence of a PCR fragmentcontaining a primer sequence at both terminal of Rhodococcus rhodocrousM8 SEQ ID NO: 63 β17RM-F primer SEQ ID NO: 64 β17RM-R primer SEQ ID NO:65 NH-19 primer SEQ ID NO: 66 NH-20 primer SEQ ID NO: 67 β37A-F primerSEQ ID NO: 68 β37A-R primer SEQ ID NO: 69 β37D-F primer SEQ ID NO: 70β37D-R primer SEQ ID NO: 71 β37F-F primer SEQ ID NO: 72 β37F-R primerSEQ ID NO: 73 β37I-F primer SEQ ID NO: 74 β37I-R primer SEQ ID NO: 75β37M-F primer SEQ ID NO: 76 β37M-R primer SEQ ID NO: 77 β37T-F primerSEQ ID NO: 78 β37T-R primer SEQ ID NO: 79 β37V-F primer SEQ ID NO: 80β37V-R primer SEQ ID NO: 81 predetermined amino-acid sequence to be usedin the present invention SEQ ID NO: 82 amino-acid sequence of β subunitrelated to the present invention SEQ ID NO: 83 α83A-F primer SEQ ID NO:84 α83A-R primer SEQ ID NO: 85 α83C-F primer SEQ ID NO: 86 α83C-R primerSEQ ID NO: 87 α83D-F primer SEQ ID NO: 88 α83D-R primer SEQ ID NO: 89α83E-F primer SEQ ID NO: 90 α83E-R primer SEQ ID NO: 91 α83F-F primerSEQ ID NO: 92 α83F-R primer SEQ ID NO: 93 α83G-F primer SEQ ID NO: 94α83G-R primer SEQ ID NO: 95 α83H-F primer SEQ ID NO: 96 α83H-R primerSEQ ID NO: 97 α83M-F primer SEQ ID NO: 98 α83M-R primer SEQ ID NO: 99α83P-F primer SEQ ID NO: 100 α83P-R primer SEQ ID NO: 101 α83S-F primerSEQ ID NO: 102 α83S-R primer SEQ ID NO: 103 α83T-F primer SEQ ID NO: 104α83T-R primer SEQ ID NO: 105 amino-acid sequence of α subunit inRhodococcus rhodocrous M8 (SU 1731814) SEQ ID NO: 106 amino-acidsequence of α subunit in Rhodococcus ruber TH SEQ ID NO: 107 amino-acidsequence of α subunit in Rhodococcus pyridinivorans MW33 (VKM Ac-1515D)SEQ ID NO: 108 amino-acid sequence of α subunit in Rhodococcuspyridinivorans S85-2 SEQ ID NO: 109 amino-acid sequence of α subunit inNocardia sp.JBRs SEQ ID NO: 110 amino-acid sequence of α subunit inNocardia sp.YS-2002 SEQ ID NO: 111 amino-acid sequence of α subunit inuncultured bacterium BD2 SEQ ID NO: 112 amino-acid sequence of α subunitin uncultured bacterium SP1 SEQ ID NO: 113 amino-acid sequence of αsubunit in Pseudonocardia thermophila JCM 3095 SEQ ID NO: 114 amino-acidsequence of α subunit in Rhodococcus rhodocrous Cr4 SEQ ID NO: 115 M8-1primer SEQ ID NO: 116 M8-2 primer SEQ ID NO: 117 amino-acid sequence ina cysteine cluster of α subunit in iron-containing nitrile hydratase SEQID NO: 118 amino-acid sequence in cysteine cluster of α subunit incobalt-containing nitrile hydratase SEQ ID NO: 119 predeterminedamino-acid sequence to be used in the present invention SEQ ID NO: 120amino-acid sequence of α subunit related to the present invention SEQ IDNO: 121 amino-acid sequence of α subunit in Rhodococcus pyridinivoransMS-38 SEQ ID NO: 122 amino-acid sequence of α subunit in Rhodococcusrhodocrous ATCC 39384 SEQ ID NO: 123 amino-acid sequence of α subunit inSinorhizobium medicae WSM419 SEQ ID NO: 124 amino-acid sequence of αsubunit in Geobacillus thermoglucosidasius Q6 SEQ ID NO: 125 amino-acidsequence of α subunit in Comamonas testosterone SEQ ID NO: 126amino-acid sequence of α subunit in Rhodococcus ruber RH (CN 101463358)SEQ ID NO: 127 α83N-F primer SEQ ID NO: 128 α83N-R primer SEQ ID NO: 129α82RM-F primer SEQ ID NO: 130 α82RM-R primer SEQ ID NO: 131 amino-acidsequence of α subunit related to the present invention SEQ ID NO: 132predetermined amino-acid sequence to be used in the present inventionSEQ ID NO: 133 α85RM-F primer SEQ ID NO: 134 α85RM-R primer SEQ ID NO:135 amino-acid sequence of α subunit related to the present inventionSEQ ID NO: 136 predetermined amino-acid sequence to be used in thepresent invention

What is claimed is:
 1. A modified Rhodococcus bacterial or Nocardiabacterial nitrile hydratase, comprising in the αsubunit, an amino-acidsequence as shown in SEQ ID NO: 136: (SEQ ID NO: 136)AX₁X₂X₃X₄GX₅X₆GX₇QX₈X₉,

wherein A is alanine, G is glycine, Q is glutamine, wherein X₁ is M(methionine), X₂ is A (alanine), X₃ is S (serine), X₄ is L (leucine), X₅is Y (tyrosine), X₆ is A (alanine), X₇ is E (glutamic acid), and X₈ is A(alanine), and X₉ is substituted with an amino acid different from thatin a wild type.
 2. The modified Rhodococcus bacterial or Nocardiabacterial nitrile hydratase according to claim 1, wherein X₉ is an aminoacid selected from the group consisting of cysteine, glutamic acid,phenylalanine, isoleucine, asparagine, glutamine, serine and tyrosine.3. The modified Rhodococcus bacterial or Nocardia bacterial nitrilehydratase according to claim 1, further comprising an amino-acidsequence as shown in SEQ ID NO: 135 comprising the amino-acid sequenceas shown in SEQ ID NO:
 136. 4. A DNA encoding the modified Rhodococcusbacterial or Nocardia bacterial nitrile hydratase according to claim 1.5. A recombinant vector, comprising the DNA according to claim
 4. 6. Atransformant, comprising the recombinant vector according to claim
 5. 7.A nitrile hydratase collected from a culture obtained by incubating thetransformant according to claim
 6. 8. A method for producing a nitrilehydratase, the method comprising: incubating the transformant accordingto claim 6; and collecting the nitrite hydratase from the obtainedculture.
 9. A method for producing an amide compound, the methodcomprising contacting a nitrile compound with a culture, or a processedproduct of the culture, obtained by incubating the nitrite hydrataseaccording to claim 1.